專利名稱：關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體的制作方法
技術(shù)領(lǐng)域：
本發(fā)明屬于生物工程技術(shù)領(lǐng)域，涉及一種用克隆關(guān)中奶山羊酪蛋白基因啟動子區(qū)構(gòu)建的能夠使目的蛋白在山羊乳腺中特異性表達(dá)并分泌到山羊乳汁中的表達(dá)載體。
背景技術(shù)：
(一)動物乳腺生物反應(yīng)器的發(fā)展歷史與研究現(xiàn)狀1980年，Gordon[1]采用顯微注射方法將重組DNA導(dǎo)入小鼠受精卵，首次獲得了帶有外源基因的轉(zhuǎn)基因小鼠，這是人類首次對哺乳動物的遺傳信息進(jìn)行人工改造的嘗試。1982年，Palmiter等[2]將大鼠生產(chǎn)激素基因注射到小鼠受精卵中，首次獲得了比普通小鼠大得多的“超級小鼠”，并提出可以從轉(zhuǎn)基因動物中提純有價值藥用蛋白的設(shè)想。“超級小鼠”誕生這一事實表明，通過性細(xì)胞的基因操作可以將外源基因?qū)氩溉閯游锏幕蚪M內(nèi)，并使其得以有效地表達(dá)，從而產(chǎn)生相應(yīng)的生理效應(yīng)。這一研究結(jié)果同時標(biāo)志著生命科學(xué)中一個嶄新研究體系一轉(zhuǎn)基因動物體系的形成。
轉(zhuǎn)基因動物被認(rèn)為是遺傳學(xué)中繼基因連鎖分析、體細(xì)胞遺傳和DNA重組之后的第四代現(xiàn)代生物技術(shù)。在“超級小鼠”誕生后的二十多年里，轉(zhuǎn)基因動物研究得到了飛速的發(fā)展。目前，轉(zhuǎn)基因小鼠制備技術(shù)已經(jīng)很成熟，并且在生物領(lǐng)域得到了廣泛的應(yīng)用，對生物科學(xué)與技術(shù)的發(fā)展發(fā)揮了巨大的推動作用。80年代以后，動物轉(zhuǎn)基因技術(shù)逐漸被應(yīng)用于家兔、家禽、山羊、綿羊、豬、牛等其它動物，目前已有數(shù)十種外源基因在上述動物中獲得表達(dá)，以轉(zhuǎn)基因動物作為生產(chǎn)活性蛋白的工廠(又稱動物生物反應(yīng)器)已經(jīng)從設(shè)想變?yōu)楝F(xiàn)實，并顯示出巨大的經(jīng)濟(jì)潛力，被譽(yù)為二十一世紀(jì)的黃金產(chǎn)業(yè)。
在動物生物反應(yīng)器研究中，研究進(jìn)展最快、開發(fā)前景最好的是動物乳腺生物反應(yīng)器。其基本原理是將具有重要開發(fā)價值的目的基因與乳蛋白基因表達(dá)調(diào)控序列融合，構(gòu)建成乳腺特異(定點(diǎn))表達(dá)基因構(gòu)件，將此基因構(gòu)件導(dǎo)入動物胚胎后獲得轉(zhuǎn)基因動物后代。當(dāng)這些動物產(chǎn)乳時，外源基因在泌乳激素的誘導(dǎo)和乳蛋白基因調(diào)控序列的指導(dǎo)下在乳腺中獲得表達(dá)，表達(dá)水平可以接近或超過正常乳蛋白的含量。因此，這種轉(zhuǎn)基因動物的乳腺猶如生產(chǎn)活性蛋白的天然工廠，只要給動物喂以飼料，就能從其乳汁中源源不斷地獲得具有生物活性的貴重蛋白。
自從重組DNA技術(shù)問世以來，人類已經(jīng)建立了許多基因工程表達(dá)系統(tǒng)來生產(chǎn)藥用蛋白。雖然這些表達(dá)系統(tǒng)都有其各自的優(yōu)點(diǎn)，但都無法與動物乳腺生物反應(yīng)器相比。目前使用最廣的是原核微生物基因工程表達(dá)系統(tǒng)，雖然其工藝流程相對簡單、研制周期較短、成本較低，其技術(shù)也相當(dāng)成熟，并已有相當(dāng)數(shù)量的基因工程產(chǎn)品造福人類，但由于原核細(xì)胞不能對真核蛋白進(jìn)行有效的加工、折疊和修飾，復(fù)雜的真核基因表達(dá)產(chǎn)物往往沒有生物活性，或以不溶性的包涵體形式存在，需經(jīng)過復(fù)雜的變性和復(fù)性過程才能獲得有用產(chǎn)品。目前研究和使用比較多的基因工程另一類系統(tǒng)是動物細(xì)胞表達(dá)系統(tǒng)，雖然其表達(dá)產(chǎn)物的活性較高，但細(xì)胞大規(guī)模培養(yǎng)的技術(shù)和條件要求苛刻，表達(dá)水平也不高，價格昂貴，故其實際應(yīng)用價值受到限制。
轉(zhuǎn)基因動物乳腺生物反應(yīng)器不僅具有上述表達(dá)系統(tǒng)的優(yōu)點(diǎn)，還可克服它們的缺點(diǎn)和不足，哺乳動物的乳腺表達(dá)系統(tǒng)具有以下優(yōu)點(diǎn)(1)在不損害動物本身健康的情況下，可方便地從乳汁中大量收集目的基因的表達(dá)產(chǎn)物。(2)動物具有較高的繁殖能力，可使大規(guī)模生產(chǎn)成為可能，且具有生產(chǎn)成本低的優(yōu)點(diǎn)。(3)乳腺上皮細(xì)胞對表達(dá)的蛋白具有完善的翻譯后加工的能力，如糖基化、磷酸化和羧基化等，從而使表達(dá)的蛋白具有與天然蛋白相似的功能。(4)正常哺乳動物乳汁的組成成分已研究得比較清楚，這樣便于目的基因表達(dá)產(chǎn)物的分離提純。哺乳動物乳汁中蛋白含量為30～35克/升，一頭奶牛每天可以產(chǎn)奶蛋白1000克，一只奶山羊可產(chǎn)奶蛋白200克，而外源基因在動物乳腺中的表達(dá)水平可以接近或超過正常乳蛋白的含量[3]。
1990年，荷蘭Pharming公司(又稱PHP公司)培育成功的表達(dá)人乳鐵蛋白的轉(zhuǎn)基因牛，每升牛奶中含有人乳鐵蛋白1克。乳鐵蛋白不僅能夠促進(jìn)嬰兒對鐵的吸收，而且能夠提高嬰兒免疫力，抵抗消化道疾病的感染，是母乳的良好替代品。3頭轉(zhuǎn)基因奶牛年產(chǎn)牛奶10噸，價值50億美元。最近，荷蘭科學(xué)家又成功培育了含有促紅細(xì)胞生成素(EPO)的轉(zhuǎn)基因牛。1991年，英國愛丁堡PPL制藥公司培育成功表達(dá)α1-抗胰蛋白酶(AAT)的轉(zhuǎn)基因綿羊[4]。具有抑制彈性蛋白酶的活性，主要用于治療囊性纖維化(CF)和肺氣腫。這種轉(zhuǎn)基因綿羊奶中的AAT含量高達(dá)35克/升，從中提純的基因工程藥物已通過II期臨床試驗，并進(jìn)入美國市場。目前，該公司已經(jīng)培育出產(chǎn)生AAT的轉(zhuǎn)基因羊200多只，正在用轉(zhuǎn)基因羊和牛乳腺生物反應(yīng)器表達(dá)的目標(biāo)產(chǎn)品近20種。美國Genzyme Transgene公司(GTC)與日本的SomitomoMetals合作，共同開發(fā)其領(lǐng)先產(chǎn)品凝血酶原III，轉(zhuǎn)基因山羊乳中重組產(chǎn)物的表達(dá)量為4g/L，目前已經(jīng)進(jìn)入臨床試驗[3]，部分基因產(chǎn)品見表一。
表一從轉(zhuǎn)基因動物奶液中分離純化目的蛋白
Clark A J，Bessos H，Bishop J O et al.Bio/Technology，1989，7487～492[14]Wright G，Binicda A，Udell M.J Chem Tech Biotechnol，1994，591I0[15]Harris D P，Andrens A T，Wright G et al.Bioseparation，1997，7(1)31～37[16]Denman J，Hayes M，Oday C dt al.Bio/Technology，1991，9839～843[17]Van Cott K E，Williams B，Velandcr W H et al.J Mol Recognit，1996，9(5～6)407～414[18]Dalton J C，BruLey D F，Kang K A et al.Adv Exp Med Biol，1997，411419～428[19]Degener A，BeLew M，Velander W H et al.J Chromatography，1998，13125～137 Wright G，Carver A，Cottom D et al，Bio/Technology.1991，9830～834[10]Paleyanda R K，Velander W H，Lcc T K et al.Nat Biotechnol，1997，1597I～975英國、美國、荷蘭等國家的一些公司利用生物反應(yīng)器生產(chǎn)抗凝血III、人乳鐵蛋白、EPO、干擾素等也已進(jìn)入臨床實驗階段。英國羅斯林研究所利用轉(zhuǎn)基因羊，批量生產(chǎn)治療肺氣腫的血友病的羊奶。我國已獲得轉(zhuǎn)人血清白蛋白、胰島素、干擾素等基因的牛、羊和兔。
中國農(nóng)業(yè)大學(xué)教授李寧等采用顯微注射法將外源人MAAT(修正的人抗胰蛋白酶)基因?qū)肷窖蛟似谂咛?nèi)，擬從山羊的奶中，提取人藥用蛋白，研發(fā)含有人保健蛋白的營養(yǎng)制品和生物藥品。有關(guān)專家認(rèn)為，獲得人基因轉(zhuǎn)基因羊，標(biāo)志著我國轉(zhuǎn)基因技術(shù)進(jìn)入了新的階段，為利用動物乳腺生物反應(yīng)器生產(chǎn)生物藥品探索出了又一個新的途徑。國外經(jīng)濟(jì)學(xué)家預(yù)測，大約10年后，轉(zhuǎn)基因動物生產(chǎn)的生物制品就會鼎足于國際市場，單是藥物的年銷售額就將超過250億美元。
(二)乳腺特異性表達(dá)的基因調(diào)控研究動物乳腺特異表達(dá)載體的構(gòu)建及其表達(dá)水平的高低在很大程度上取決于對乳蛋白基因表達(dá)調(diào)控機(jī)理的認(rèn)識。目前，已克隆出許多乳蛋白基因，部分乳蛋白基因及其調(diào)控序列的核昔酸己被發(fā)表。用于轉(zhuǎn)基因動物乳腺定位表達(dá)的調(diào)控元件主要有以下四類第一類β-乳球蛋白(BLG)基因調(diào)控元件。Simons等將綿羊的BLG基因轉(zhuǎn)入小鼠，綿羊的β-乳球蛋白在小鼠乳腺中特異性表達(dá)。其奶液中含量可達(dá)23g/L[8]。
第二類酪蛋白基因調(diào)控序列。常用牛αS1-酪蛋白基因和羊β-酪蛋白基因的調(diào)控序列。如αS1-酪蛋白基因調(diào)控序列指導(dǎo)的人白介素-2基因已在轉(zhuǎn)基因兔奶液中成功表達(dá)[9]。
第三類乳清酸蛋白(WAP)基因調(diào)控序列。WAP是嚙齒類動物奶液中的主要蛋白質(zhì)，在家畜奶液中沒有WAP的存在。但WAP基因調(diào)控序列可以指導(dǎo)外源基因在家畜奶液中表達(dá)[10]。
第四類乳清白蛋白基因調(diào)控序列。
盡管不同動物乳汁中乳蛋白的含量不同，但含量最高的都是酪蛋白，在牛奶和羊奶中β-酪蛋白分別約占總?cè)榈鞍椎?7％和50％以上(Provot et al 1995)，表明β-酪蛋白啟動子活性很強(qiáng)，它能啟動外源基因在轉(zhuǎn)基因動物的乳腺組織中表達(dá)(vi-vito)。因此酪蛋白基因是主要的研究對象。
Gordon K.et al.Genetic transformation of mouse embryos by microinjection of purifiedDNA.PNAS.1980，777380-7348[2]Palmiter RD et al.Dramatic growth of mice that develop from eggs microin-jected withmetallothionein-growth hormone fusiongenes Nature.1982，300611[3]薛京倫 盧大儒 乳腺生物反應(yīng)器的研究現(xiàn)狀，生物技術(shù)通報.1998，317-21[4]Wright G，Carver A et al.High level expression of active human alpha-l-antitrypsin inthe milk of transgenic sheep.Bio/Technology.1991，9830-834- --- -[8]Simons J P，Mccienaghan M，Clark A J.Natare，1987，328530～532[9]Buhler T，Bruyere T B，Went D F et al.Bio/Technology，1990，8140～143[10]Paleyanda R K，Velander W H，Lee T K et al.Nat Biotechnol，1997，15971～975(三)、與本發(fā)明相關(guān)技術(shù)上海市兒童醫(yī)院上海醫(yī)學(xué)遺傳研究所曾溢滔教授領(lǐng)導(dǎo)的研究組在遺傳學(xué)報29(3)；206-211，2002發(fā)表關(guān)于《山羊β-酪蛋白基因啟動子指導(dǎo)的轉(zhuǎn)基因小鼠乳汁高效表達(dá)人凝血因子IX》一文中，應(yīng)用β-酪蛋白基因啟動子6.7kb指導(dǎo)外源基因表達(dá)，其所構(gòu)建的山羊β-酪蛋白基因啟動子序列在保留原有近端成分外，還保留了上游調(diào)控區(qū)和β-酪蛋白第一內(nèi)含子、第一外顯子和第二外顯子。但將目的蛋白插入載體進(jìn)行分泌性表達(dá)，其效果不理想，原因和結(jié)果都需要經(jīng)過再試驗。

發(fā)明內(nèi)容
針對上述現(xiàn)有技術(shù)中存在的不足，本發(fā)明的目的在于提供一種以西北楊凌特有的高產(chǎn)奶山羊的乳腺組織作為β-酪蛋白基因啟動子序列來源的表達(dá)載體，此表達(dá)載體能夠用于建立轉(zhuǎn)基因山羊乳腺生物反應(yīng)器，使用基因工程手段所構(gòu)建的目的蛋白特異性地在山羊乳腺中表達(dá)、并分泌到山羊乳汁中，以供制備生產(chǎn)目的蛋白。
本發(fā)明關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體，具有商售質(zhì)粒載體和β-酪蛋白基因啟動子及其周圍的調(diào)控序列以及第一內(nèi)含子、第一外顯子和第二外顯子所構(gòu)成的β-酪蛋白基因啟動子區(qū)域，其特征在于(1)、所述的β-酪蛋白基因啟動子區(qū)域序列還包括在第二外顯子末端引入作為信號肽的限制性核酸內(nèi)切酶SgfI位點(diǎn)序列；該啟動區(qū)域序列為-4359～+2106bp(basepair)，全長6465bp；
(2)、所述的β-酪蛋白基因啟動子是以西北楊凌特有的高產(chǎn)關(guān)中奶山羊的乳腺組織作為來源，從中提取基因組DNA作為模板，設(shè)計合成引物，用高保真的DNA聚合酶進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)(PCR)，獲得PCR產(chǎn)物，經(jīng)過拼接、克隆和測定序列而得；(3)、所述的關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體是將含關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列6465bp通過限制性內(nèi)切酶插入商售的質(zhì)粒載體中而得。關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列全長6465bp如下所示1 AGATGATTTT GCAACCCCCT GCCTCAGGAG ACACTGGGAA ATTTCCTGAG ACATTTTTGATCTACTAAAA CGTTGGGGGA CGGAGTCCTC TGTGACCCTT TAAAGGACTC TGTAAAAACT61 TTCCAAAAGC TGTGCAGTTG GTGCTTCTAC CATCTTCGTG GTAGAGGTCA AGGATGCTGCAAGGTTTTCG ACACGTCAAC CACGAAGATG GTAGAAGCAC CATCTCCAGT TCCTACGACG121 TAAACATTCT ACAACACATT AAGAAAACCC CCACAACAAA GAATTCTTCC GCCAAAAATAATTTGTAAGA TGTTGTGTAA TTCTTTTGGG GGTGTTGTTT CTTAAGAAGG CGGTTTTTAT181 TCAATAATAT GAAGGTTGAA AAATACTGGT CTAGCATGTA GTATGTGCTC AATAGCAAGGAGTTATTATA CTTCCAACTT TTTATGACCA GATCGTACAT CATACACGAG TTATCGTTCC241 AGAGAAAAGA AAGCCTTCCT CACTGATTAA TGCAAAGAAA TAGAGGAAAA CAATAGAATGTCTCTTTTCT TTCGGAAGGA GTGACTAATT ACGTTTCTTT ATCTCCTTTT GTTATCTTAC301 GGAAAGACTA GAGAGCTCTT CAAGCAAATT AGAGATATCA AGGGAACATT TCACGCAAAGCCTTTCTGAT CTCTCGAGAA GTTCGTTTAA TCTCTATAGT TCCCTTGTAA AGTGCGTTTC361 ATGGGCACAA TAAAGGACAG AAATTTTATG GAGGAGTTGC TGATGGAGAG GGAGGCCTGGTACCCGTGTT ATTTCCTGTC TTTAAAATAC CTCCTCAACG ACTACCTCTC CCTCCGGACC421 CGTGCTGCGA TTCCTGGGGT CGCAAAGAGT CGGACACAAC TGAGCGACTG AATTGAACTGGCACGACGCT AAGGACCCCA GCGTTTCTCA GCCTGTGTTG ACTCGCTGAC TTAACTTGAC481 AACTGAACTG GACAAAGCAG AAGATATTAA GAAGAGGTGG TAAGAATACA CAGAAGAACATTGACTTGAC CTGTTTCGTC TTCTATAATT CTTCTCCACC ATTCTTATGT GTCTTCTTGT541 ATATAAAAAA GATCTTCATG ACCCAGATAA CCACGATGAT GTGATCACTC ACCTAGAGCCTATATTTTTT CTAGAAGTAC TGGGTCTATT GGTGCTACTA CACTAGTGAG TGGATCTCGG601 AGACACCCTG GAATGCAAAG TCAAACGGCC TTAGAAAGCC TCACTATGAA CAAAGCTAGTTCTGTGGGAC CTTACGTTTC AGTTTGCCGG AATCTTTCGG AGTGATACTT GTTTCGATCA661 GGAGGTAATG GAATTCCAGT TGAGCTATTT CAAATCTTAA AAGGTGATGC TGTGAAAGTGCCTCCATTAC CTTAAGGTCA ACTCGATAAA GTTTAGAATT TTCCACTACG ACACTTTCAC721 CTGCACTCAA TATGTCAGCA AATTTGGAAA ACTCAGCAGT GGCCACAGGA CTGCCACAATGACGTGAGTT ATACAGTCGT TTAAACCTTT TGAGTCGTCA CCGGTGTCCT GACGGTGTTA781 CCCAAAGAAA AGCAATGACA AAGAATGTTC AAACACCCAC ATGATTGCAC TCATCTCACAGGGTTTCTTT TCGTTACTGT TTCTTACAAG TTTGTGGGTG TACTAACGTG AGTAGAGTGT841 TGCTAGCAAA ATAACTCTCA AAATTCTCCA AGCCAGGCTC CAACAGTACG TGGACCATGAACGATCGTTT TATTGAGAGT TTTAAGAGGT TCGGTCCGAG GTTGTCATGC ACCTGGTACT901 ACTTCCAGAT GTTCAAGCTG GATTTAGAAA AGGCAGAGGA ACCAGAGATC AAATTGCCAATGAAGGTCTA CAAGTTCGAC CTAAATCTTT TCCGTCTCCT TGGTCTCTAG TTTAACGGTT961 CATCCATTGG ATCATCAAAA AAGCACGAGA GTTCCAGAAA AACATCTGCT TTATTGACTAGTAGGTAACC TAGTAGTTTT TTCGTGCTCT CAAGGTCTTT TTGTAGACGA AATAACTGAT1021 CGCTAAAGCC TTTGATTGTG TGGATCACAA TAAACTGTGG AAAATTCTTC AAGAGATGGGGCGATTTCGG AAACTAACAC ACCTAGTGTT ATTTGACACC TTTTAAGAAG TTCTCTACCC1081 AATACCAGAC CACTTTACCT GCCTCCTGAG AAATCTGTAT ACAGGTCCAG AAGCAGCAGTTTATGGTCTG GTGAAATGGA CGGAGGACTC TTTAGACATA TGTCCAGGTC TTCGTCGTCA1141 TAGAACTGGA CATGGAACAA CAGACTGGTT CCAAACTGCG AAAGGGGTAC ATCAAGGAATATCTTGACCT GTACCTTGTT GTCTGACCAA GGTTTGACGC TTTCCCCATG TAGTTCCTTA1201 ATTCATTGGA AGGATTGATG CTGAAGCTGA AACTCCTATA CTTTGGCCAC CTAATGTGAATAAGTAACCT TCCTAACTAC GACTTCGACT TTGAGGATAT GAAACCGGTG GATTACACTT1261 GATCTGACTC ATTGGAAAAG ACTCCAATGC TGGGAAAGAT TGAAGGCAGG AGAAGAGGATCTAGACTGAG TAACCTTTTC TGAGGTTACG ACCCTTTCTA ACTTCCGTCC TCTTCTCCTA1321 GACAGAGGAT GAGATGGTTG GATGGGATCA CTGACTCAAT GGACATGAGT TTGAGTAAGCCTGTCTCCTA CTCTACCAAC CTACCCTAGT GACTGAGTTA CCTGTACTCA AACTCATTCG
1381 TCCAGGGGTT GGTGGTGGAC AGGAAAGCCT GGCGTGCTGC AGTCCACAAG GTCACAAAGAAGGTCCCCAA CCACCACCTG TCCTTTCGGA CCGCACGACG TCAGGTGTTC CAGTGTTTCT1441 TTCGGACATG ACTGAGTGAC TGAACTGATA CTGATGTGCT CAACAAATGT ATCTTGAACTAAGCCTGTAC TGACTCACTG ACTTGACTAT GACTACACGA GTTGTTTACA TAGAACTTGA1501 TGTGTGAAGT TCTATGGTCA CATGTAAAGG AAGAATAATC AGGATTAGCT GTGTGTCTTAACACACTTCA AGATACCAGT GTACATTTCC TTCTTATTAG TCCTAATCGA CACACAGAAT1561 GGAATCAGGG TTCTGAGTTT TATGTGTTCA TAGTATCTGC TGGTTCACAA AACATTTTTCCCTTAGTCCC AAGACTCAAA ATACACAAGT ATCATAGACG ACCAAGTGTT TTGTAAAAAG1621 TTATTCTCTG GTTCTTGATT TACTTTATAA AGTAATCTTA ATAGTTATAC TTCACATAGAAATAAGAGAC CAAGAACTAA ATGAAATATT TCATTAGAAT TATCAATATG AAGTGTATCT1681 TACGAAATTA TTATATTTGG ATAATCTCAT GGAAAGGATT AAATACTCCA TCTATTACGAATGCTTTAAT AATATAAACC TATTAGAGTA CCTTTCCTAA TTTATGAGGT AGATAATGCT1741 GTAATGCTGA ACTATCTACT CCTACCTAAT AATTTGTCAG AATTCACTAA TTCTGTGTTACATTACGACT TGATAGATGA GGATGGATTA TTAAACAGTC TTAAGTGATT AAGACACAAT1801 TATTGTTTCT AAATCTGAAT CATTATATGA ATCCTCAGTA TTTTGTTTTC CTTCCTCTATATAACAAAGA TTTAGACTTA GTAATATACT TAGGAGTCAT AAAACAAAAG GAAGGAGATA1861 ATTTTGGAAT TTATTAAACA GTGCTTCAAA TAATTTTTAG GAAACTGAAG TTTTTAGTAATAAAACCTTA AATAATTTGT CACGAAGTTT ATTAAAAATC CTTTGACTTC AAAAATCATT1921 CAGCTCTATC TCTAAATAGC TTTAGTATCT TGAAAAAGTA ATACAAATTC TCACATCCTTGTCGAGATAG AGATTTATCG AAATCATAGA ACTTTTTCAT TATGTTTAAG AGTGTAGGAA1981 AATTTCCTCT TCTCTAAAAT ATCTTTAAAA TATTCTATGA ATGATATCTC TTAATATTTATTAAAGGAGA AGAGATTTTA TAGAAATTTT ATAAGATACT TACTATAGAG AATTATAAAT2041 TTTTTTTGGC AATCCAACAC AGCTTATGGG ATCTTAGTTC CCCAGTGAGG GATTATATCCAAAAAAACCG TTAGGTTGTG TCGAATACCC TAGAATCAAG GGGTCACTCC CTAATATAGG2101 ATGCCAACTG CAGTGAAAGT ACAAAATCCT AAACTGGACT CACCAGGGAT TTCCCAATATTACGGTTGAC GTCACTTTCA TGTTTTAGGA TTTGACCTGA GTGGTCCCTA AAGGGTTATA2161 CTCCTCTAGT TCTTATTTCT GAATATTTTT GGTCCCTTTA TTGTACTCTT CATCCAACTTGAGGAGATCA AGAATAAAGA CTTATAAAAA CCAGGGAAAT AACATGAGAA GTAGGTTGAA2221 TTCTATTGAT TTCTTTCTTG AGGTTATTAT TTACTTGGTT TCAGTTAGAA ATATATGCAAAAGATAACTA AAGAAAGAAC TCCAATAATA AATGAACCAA AGTCAATCTT TATATACGTT2281 ATCTCAGGAC TGCATATTTC AGATTCATTG GCCAATATGG GAAAAAACCT TTGGCTGAACTAGAGTCCTG ACGTATAAAG TCTAAGTAAC CGGTTATACC CTTTTTTGGA AACCGACTTG2341 AAATCATGCT TATAAAAAAT AGTACTAGAG CATCCTACTT TGACTATATC TTGCTCCTCATTTAGTACGA ATATTTTTTA TCATGATCTC GTAGGATGAA ACTGATATAG AACGAGGAGT2401 TTCAGGGTTA TCTAATACAA TTTCCCCACA TGAAATTCTT TTGCATTATA AAAATGGAAGAAGTCCCAAT AGATTATGTT AAAGGGGTGT ACTTTAAGAA AACGTAATAT TTTTACCTTC2461 CTCTTAGGTA ACATTGCAAA AATTCGAGTT GCTCATATGG CACTTTGCTT CTTACTGGTCGAGAATCCAT TGTAACGTTT TTAAGCTCAA CGAGTATACC GTGAAACGAA GAATGACCAG2521 ATTGTGTTCT GAGGCTTACC TGGACAGGTG GTACCTGATG TCATCTTAAA TTGCTGGCTTTAACACAAGA CTCCGAATGG ACCTGTCCAC CATGGACTAC AGTAGAATTT AACGACCGAA2581 TTTGATTTTC CATTGGACAA GCTTCTTTCT TTAGTATATT GTTAAGGATT TCCTTGATCAAAACTAAAAG GTAACCTGTT CGAAGAAAGA AATCATATAA CAATTCCTAA AGGAACTAGT2641 AGATTTTACC TACTTTTCTG GTCCAATTGG TGAGAGACAG TCATAAGGAA ATGCTGTGTTTCTAAAATGG ATGAAAAGAC CAGGTTAACC ACTCTCTGTC AGTATTCCTT TACGACACAA2701 TATTGCACAA TATGTAAAGC ATCTTCCTGA GAAAATAAAA GGGAAATGTT GAATGGGAAGATAACGTGTT ATACATTTCG TAGAAGGACT CTTTTATTTT CCCTTTACAA CTTACCCTTC2761 GATATGCTTT CTTTTGTATT CCTTTTCTGA GAAATCAAAC TTTTTCACCT GTGGCCTTGGCTATACGAAA GAAAACATAA GGAAAAGACT CTTTAGTTTG AAAAAGTGGA CACCGGAACC2821 CCACCAAAAG CTAACAAATA AAGGCATATG AAGTAGCCAA GGCCTTTTCT AGTTATATCTGGTGGTTTTC GATTGTTTAT TTCCGTATAC TTCATCGGTT CCGGAAAAGA TCAATATAGA2881 ATAACACTGA GTTCATTTCA TCATTTATTT TCCTGACTTC CTCCTGGGTC CATATGAGCATATTGTGACT CAAGTAAAGT AGTAAATAAA AGGACTGAAG GAGGACCCAG GTATACTCGT2941 GTCTTAGAAT GAATATTAGC TGAATAATCC AAATACATAG TAGATGTTGA TTTGGGTTTTCAGAATCTTA CTTATAATCG ACTTATTAGG TTTATGTATC ATCTACAACT AAACCCAAAA3001 CTAAGCAATC CAAGACTTGT ATGACAGTAA GATGTATTAC CATCCAACAC ACATCTCAGCGATTCGTTAG GTTCTGAACA TACTGTCATT CTACATAATG GTAGGTTGTG TGTAGAGTCG3061 ATGATATAAA TGCAAGGTAT ATTGTGAAGA AAAATTTTTA ATTATGTCAA AGTGCTTACTTACTATATTT ACGTTCCATA TAACACTTCT TTTTAAAAAT TAATACAGTT TCACGAATGA
3121 TTAGAAGGTC ATCTATCTGT CCCAAAGCTG TGAATATATA TATTGAAGGT AATGAATAGAAATCTTCCAG TAGATAGACA GGGTTTCGAC ACTTATATAT ATAACTTCCA TTACTTATCT3181 TGAAGCTAAC CTTGTAAAAA TGAGTAGTGT GAAATACAAC TACAATTATG AACATCTGTCACTTCGATTG GAACATTTTT ACTCATCACA CTTTATGTTG ATGTTAATAC TTGTAGACAG3241 ACTAAAGAGG CAAAGAAACT TGAAGATTGC TTTTGCAAAT GGGCTCCTAT TAATAAAAAGTGATTTCTCC GTTTCTTTGA ACTTCTAACG AAAACGTTTA CCCGAGGATA ATTATTTTTC3301 TACTTTTGAG GTCTGGCTCA GACTCTATTG TAGTACTTAG GGTAAGACCC TCCTCCTGTAATGAAAACTC CAGACCGAGT CTGAGATAAC ATCATGAATC CCATTCTGGG AGGAGGACAT3361 TGGGCTTTCA TTTTCTTTCT TGCTTCCCTC ATTTGCCCTT CCATGAATAC TAGCTGATAAACCCGAAAGT AAAAGAAAGA ACGAAGGGAG TAAACGGGAA GGTACTTATG ATCGACTATT3421 ACATTGACTA TAAAAGATAT GAGGCCAAAC TTGAGCTGTC CCATTTTAAT AAATCTGTATTGTAACTGAT ATTTTCTATA CTCCGGTTTG AACTCGACAG GGTAAAATTA TTTAGACATA3481 AAATAATATT TGTTCTACAA AAGTATTATC TAAATAAATG TTACTTTCTG TCTTAAAATCTTTATTATAA ACAAGATGTT TTCATAATAG ATTTATTTAC AATGAAAGAC AGAATTTTAG3541 CCTCAACAAA TCCCCACTAT CTAGAGAATA AGATTGACAT TCCCTGGAAT CACAGCATGCGGAGTTGTTT AGGGGTGATA GATCTCTTAT TCTAACTGTA AGGGACCTTA GTGTCGTACG3601 TTTGTCTGCC ATTATCTGAC CCCTTTCTCT TTCTCTCTTC TCACCTCCAT CTACTCCTTTAAACAGACGG TAATAGACTG GGGAAAGAGA AAGAGAGAAG AGTGGAGGTA GATGAGGAAA3661 TTCCTTGCAA TTCATGACCC AGATTCACTG TTTGATTTGG CTTGCATGTG TGTGTGCTGAAAGGAACGTT AAGTACTGGG TCTAAGTGAC AAACTAAACC GAACGTACAC ACACACGACT3721 GTTGCGTCTG ACTGTTATCA ACCCCATGAA TGATAGTCCA CCAGGCTCTA CTGTCCATGACAACGCAGAC TGACAATAGT TGGGGTACTT ACTATCAGGT GGTCCGAGAT GACAGGTACT3781 AATTTTCCAG TCAAGAATAC TGGAGTGGAT TGCATTTCCT ACTCCATTTG ATTAATTTAGTTAAAAGGTC AGTTCTTATG ACCTCACCTA ACGTAAAGGA TGAGGTAAAC TAATTAAATC3841 TGACTTTTAA ATTTCTTTTT CCATATTCGG GAGCCTATTC TTCCTTTTTA GTCTATACTCACTGAAAATT TAAAGAAAAA GGTATAAGCC CTCGGATAAG AAGGAAAAAT CAGATATGAG3901 TCTTCACTCT TCAGGTCTAA GGTATCATCG TGTGCTTGTT AGCTTGTTAC TTTCTCCATTAGAAGTGAGA AGTCCAGATT CCATAGTAGC ACACGAACAA TCGAACAATG AAAGAGGTAA3961 ATAGCTTAAG CACTAACAAC TGTTCAGGTT GGCATGAAAT TGTGTTCTTT GTGTGGCCTGTATCGAATTC GTGATTGTTG ACAAGTCCAA CCGTACTTTA ACACAAGAAA CACACCGGAC4021 TATATTTCTG TTGTGTATTA GAATTTACCC CAAGATCTCA AAGACCCACT GAATACTAAAATATAAAGAC AACACATAAT CTTAAATGGG GTTCTAGAGT TTCTGGGTGA CTTATGATTT4081 GAGACCTCAT TGTGGTTACA ATAATTTGGG GACTGGGCCA AAACTACCGT GCATCCCAGCCTCTGGAGTA ACACCAATGT TATTAAACCC CTGACCCGGT TTTGATGGCA CGTAGGGTCG4141 CAAGATCTGT AGCTACTGGA CAATTTCATT TCCTTTATCA GATTGTGAGT TATTCCTGTTGTTCTAGACA TCGATGACCT GTTAAAGTAA AGGAAATAGT CTAACACTCA ATAAGGACAA4201 AAAATGCTCC CCAGAATTTC TGGGGACAGA AAAATAGGAA GAATTCATTT CCTAATCATGTTTTACGAGG GGTCTTAAAG ACCCCTGTCT TTTTATCCTT CTTAAGTAAA GGATTAGTAC4261 CAGATTTCTA GGAATTCAAA TCCACTGTTG GTTTTATTTC AAACCACAAA ATTAGCATGCGTCTAAAGAT CCTTAAGTTT AGGTGACAAC CAAAATAAAG TTTGGTGTTT TAATCGTACG4321 CATTAAATAC TATATATAAA CAGCCACTAA ATCAGATCAT TATCCATTCA GCTTCTCCTTGTAATTTATG ATATATATTT GTCGGTGATT TAGTCTAGTA ATAGGTAAGT CGAAGAGGAA4381 CACTTCTTCT CCTCTACTTT GGAAAAAAGG TAAGAATCTC AGATATAATT TCAGGTGTATGTGAAGAAGA GGAGATGAAA CCTTTTTTCC ATTCTTAGAG TCTATATTAA AGTCCACATA4441 CTGCTACTCA TCTTTATTTT GGACTAGGTT AAAATGTAGA AAGAACATAA TTGCTTAAAAGACGATGAGT AGAAATAAAA CCTGATCCAA TTTTACATCT TTCTTGTATT AACGAATTTT4501 TAGATCTTAA AAATAAGGGT GTTTAAGATA AGGTTTACAC TATTTTCAGC AGATATGTTAATCTAGAATT TTTATTCCCA CAAATTCTAT TCCAAATGTG ATAAAAGTCG TCTATACAAT4561 AAAAATAGAA GTGACTATAA AGACTTGATA AAAATTATAG TGACTGCAAA TGTTTTAGGATTTTTATCTT CACTGATATT TCTGAACTAT TTTTAATATC ACTGACGTTT ACAAAATCCT4621 ATATAATAAG ATATAATAAC GGTGGTTGCT ATTTTCTTTA GCACAAGACT AGTTAACAGGTATATTATTC TATATTATTG CCACCAACGA TAAAAGAAAT CGTGTTCTGA TCAATTGTCC4681 CTGTATTAAA AGATCTTTTC TTGAATTAAA TATTTTCAAT TTGATTAAAC CTACCTCAGCGACATAATTT TCTAGAAAAG AACTTAATTT ATAAAAGTTA AACTAATTTG GATGGAGTCG4741 CATAAAGGCA AGCACATTTC ATTTATACTA TGGGGATTTG AATAATTATT ACTGAAGAAGGTATTTCCGT TCGTGTAAAG TAAATATGAT ACCCCTAAAC TTATTAATAA TGACTTCTTC4801 CTCTACCAAC AAAAAGTTTA TAGAGCTATC ATATTTAGTC AAGAGATAAA GAGGGTTGTTGAGATGGTTG TTTTTCAAAT ATCTCGATAG TATAAATCAG TTCTCTATTT CTCCCAACAA
4861 AGGATATATA TGCTATTTGA AAGGTATTTA TAAAAGAAGA GTATATTTAT CAAAATTTCTTCCTATATAT ACGATAAACT TTCCATAAAT ATTTTCTTCT CATATAAATA GTTTTAAAGA4921 CAGAACATCC AAATTTCAAG TTTATCATTT ATCTTACAAT ATTTCAAAAA TATTAAAATAGTCTTGTAGG TTTAAAGTTC AAATAGTAAA TAGAATGTTA TAAAGTTTTT ATAATTTTAT4981 GATACTGAAA TACAGAAGTA AATTAAAGAG AAAGTATTTT ACTTGGTAAA AAAATTCTAGCTATGACTTT ATGTCTTCAT TTAATTTCTC TTTCATAAAA TGAACCATTT TTTTAAGATC5041 GTTGGACAGA GAGTGCCAGG AAACAAAAAC AATGAAAAAT GTGACCTGAC AGGAATTATACAACCTGTCT CTCACGGTCC TTTGTTTTTG TTACTTTTTA CACTGGACTG TCCTTAATAT5101 GCTCAAAGTA TAGTAGTAAG TAATGAAATG GCTTAAAAAT TGGTATATAA AATGCTAGTTCGAGTTTCAT ATCATCATTC ATTACTTTAC CGAATTTTTA ACCATATATT TTACGATCAA5161 ATAAAATAAA CAAAATGCAA TAATATCCTC CCTACATGTA ATGAATTCTA GGTATTATGCTATTTTATTT GTTTTACGTT ATTATAGGAG GGATGTACAT TACTTAAGAT CCATAATACG5221 TCTTTTTGGA AGTCTTGACA ATAAAAATTT TTTTAGAAGT TTATAGGCAT CTTGAATAAAAGAAAAACCT TCAGAACTGT TATTTTTAAA AAAATCTTCA AATATCCGTA GAACTTATTT5281 GTGAAACAAA TTAAGAATTA GTATCCATGA GAAAAATATA GAACAATTTT CCTAATTTAGCACTTTGTTT AATTCTTAAT CATAGGTACT CTTTTTATAT CTTGTTAAAA GGATTAAATC5341 TTTGAAAATC TGGGATTGAA GATGTGTGTC AAGAGATGTT GGTGGCAAGA ACATTTTTTTAAACTTTTAG ACCCTAACTT CTACACACAG TTCTCTACAA CCACCGTTCT TGTAAAAAAA5401 TTCAAGAACT TATAAAAATG CAACAAAACA AACCATTTAA TACATTTTGG TCAAAATCAAAAGTTCTTGA ATATTTTTAC GTTGTTTTGT TTGGTAAATT ATGTAAAACC AGTTTTAGTT5461 TAATGTATTT TATTTTATGC TCCAAGGAGC ATAAAATTGG GGACTGGGCA AGAGAAACTGATTACATAAA ATAAAATACG AGGTTCCTCG TATTTTAACC CCTGACCCGT TCTCTTTGAC5521 ACACCCTGGT AAATTACCAA GAGATAAGTA CACAGTTCTA TGTAGAGAAA ATAAGCATAGTGTGGGACCA TTTAATGGTT CTCTATTCAT GTGTCAAGAT ACATCTCTTT TATTCGTATC5581 TGTATGATCT CTAAAATTAT GTGAGACAAA GGAGAGATGA CATTAGGCAT GTGGGGATGAACATACTAGA GATTTTAATA CACTCTGTTT CCTCTCTACT GTAATCCGTA CACCCCTACT5641 AGACTGAGTA GAGAAGAAAC AATCTAATCA GTCCAAGAAA ACATCTCGAT CAGTGGAACATCTGACTCAT CTCTTCTTTG TTAGATTAGT CAGGTTCTTT TGTAGAGCTA GTCACCTTGT5701 AATAGAAGAA ATGCTAAAAT GAAACAGAAG TCTTACTGGA AATAAAAGAT ATGCATAAGATTATCTTCTT TACGATTTTA CTTTGTCTTC AGAATGACCT TTATTTTCTA TACGTATTCT5761 CAAAAATTCA TGAAAATCAC TTAGTTTAGC AGAGAAAAGA TAAAAATAAA GTATGACCTTGTTTTTAAGT ACTTTTAGTG AATCAAATCG TCTCTTTTCT ATTTTTATTT CATACTGGAA5821 CTTCATATAC ATTGTTTGAT CATATGCACC TCAATAAAAC TGAGTCTCCA ACAGAAATGAGAAGTATATG TAACAAACTA GTATACGTGG AGTTATTTTG ACTCAGAGGT TGTCTTTACT5881 AACATTAATA TTTTGTTCAC TGCTCTAATC CCAGAATCTA AGCGATATCT GGCAATAAAATTGTAATTAT AAAACAAGTG ACGAGATTAG GGTCTTAGAT TCGCTATAGA CCGTTATTTT5941 ATAATAAATA TATATTTTTT AATAAATGAA TCAACCACTT AATTTTTCTG TAAATATCTGTATTATTTAT ATATAAAAAA TTATTTACTT AGTTGGTGAA TTAAAAAGAC ATTTATAGAC6001 TAACTTCTCT TCTGTCTTTC CAAAAACACT CATAAGTACT GTGAATGAGA TGAAAAAGAGATTGAAGAGA AGACAGAAAG GTTTTTGTGA GTATTCATGA CACTTACTCT ACTTTTTCTC6061 TGAAGTAGGA TATAGGCTGT TAGCAGAAAA CATCTGAATG GCTGGCAGTG AAACATTAACACTTCATCCT ATATCCGACA ATCGTCTTTT GTAGACTTAC CGACCGTCAC TTTGTAATTG6121 TTGAAATGTA AGATTAATGA GTAATAGTAA ATTTTAACCT TGGCCATATG ATAAAATGTTAACTTTACAT TCTAATTACT CATTATCATT TAAAATTGGA ACCGGTATAC TATTTTACAA6181 CATTAATATT TTTCTAGAAT ACAGGGCTTT TTGTTTTTGC CATGAGGTTT GCAGGATCTTGTAATTATAA AAAGATCTTA TGTCCCGAAA AACAAAAACG GTACTCCAAA CGTCCTAGAA6241 GGTTCCCTGA CCAGGGATCA AACCTGCACT CCCCTGGAAG CATGGAGTCT TGGACATTTGCCAAGGGACT GGTCCCTAGT TTGGACGTGA GGGGACCTTC GTACCTCAGA ACCTGTAAAC6301 TATTATACAC TATCTTTGGT TCCTTTTAAA GGGAAGTAAT TTTACTTAAA TAAGAAAATAATAATATGTG ATAGAAACCA AGGAAAATTT CCCTTCATTA AAATGAATTT ATTCTTTTAT6361 GATTGACAAG TAATACGCTG TTTCCTCATC TTCCCATTCA CAGGAATCGA GAGCCATGAACTAACTGTTC ATTATGCGAC AAAGGAGTAG AAGGGTAAGT GTCCTTAGCT CTCGGTACTT6421 GGTCCTCATC CTTGCCTGTC TGGTGGCTCT GGCCATTGCG ATCGCCCAGGAGTAG GAACGGACAG ACCACCGAGA CCGGTAACGC TAGCG本發(fā)明關(guān)中奶山羊β-酪蛋白啟動子經(jīng)過實驗檢測，證明具有啟動子和增強(qiáng)子活性。檢測實驗及結(jié)果如下
1、采取啟動子和增強(qiáng)子捕獲(promoter and enhancer trap)技術(shù)進(jìn)行檢測方法是使用Promega公司商售的熒光素酶報告基因載體pGL3-Basic Vector(其環(huán)狀結(jié)構(gòu)圖見圖3)，將本發(fā)明6465bp的啟動子序列插入其多克隆限制性內(nèi)切酶位點(diǎn)KpnI和BglII之間，構(gòu)建成含6465bp啟動子序列的質(zhì)粒pGL3-B65，瞬時轉(zhuǎn)染原代培養(yǎng)的山羊乳腺上皮細(xì)胞，在催乳素(prolactin)誘導(dǎo)下，報告基因—熒光素酶的合成量顯著增高。實驗數(shù)據(jù)如下

實驗結(jié)果充分說明所克隆的6465bp DNA序列具有啟動子和增強(qiáng)子活性。
2、采取啟動子捕獲(promoter trap)技術(shù)進(jìn)行檢測方法是使用Promega公司商售的熒光素酶報告基因載體pGL3-Enhancer Vector(其環(huán)狀結(jié)構(gòu)圖見附圖4)，將本發(fā)明6465bp的啟動子序列插入其多克隆限制性內(nèi)切酶位點(diǎn)KpnI和BglII之間，構(gòu)建成含6465bp的啟動子序列的質(zhì)粒pGL3-E65，瞬時轉(zhuǎn)染原代培養(yǎng)的山羊乳腺上皮細(xì)胞，在催乳素(prolactin)誘導(dǎo)下，報告基因—熒光素酶的合成量顯著增高。實驗數(shù)據(jù)如下

上述2組數(shù)據(jù)統(tǒng)計學(xué)顯著性測驗P＜0.05。實驗結(jié)果表明所克隆的6465bp DNA序列具有啟動子活性。


圖1是β-酪蛋白啟動子區(qū)DNA序列示意2是Clontech公司的商售質(zhì)粒pcDNA3.1(+/-)結(jié)構(gòu)示意3是Promega公司的商售質(zhì)粒pGL3-Basic Vector結(jié)構(gòu)示意4是Promega公司的商售質(zhì)粒pGL3-Enhancer Vector結(jié)構(gòu)示意圖其中圖1本發(fā)明所克隆構(gòu)建的關(guān)中奶山羊乳腺β-酪蛋白基因啟動區(qū)序列包含β-酪蛋白基因上游的啟動子區(qū)域，編碼乳腺β-酪蛋白基因的第一、第二外顯子和第一內(nèi)含子，以及在第二外顯子末端引入的限制性核酸內(nèi)切酶Sgf I位點(diǎn)序列，以利于外源目的基因的插入和表達(dá)。
圖2中Pcmv為CMV啟動子；BGH PA為牛生長激素基因的多聚腺苷酸序列；flori為DNA單鏈復(fù)制起點(diǎn)；SV40ori為SV40病毒復(fù)制起點(diǎn)；Neomycin為新霉素抗性基因；Ampicillin為氨芐青霉素抗性基因；SV40pA為SV40病毒多聚腺苷酸序列；PUCori為pUC質(zhì)粒復(fù)制起點(diǎn)；T7 Nhel……Pmel為多限制性內(nèi)切酶位點(diǎn)序列，是外源DNA插入的位置。本發(fā)明將所克隆的β-酪蛋白基因啟動區(qū)序列6465bp插入pcDNA3.1(-)中的外源DNA插入位置T7 Nhel……Pmel多限制性內(nèi)切酶位點(diǎn)，即得到本發(fā)明所需要的一種關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體，至限制性內(nèi)切酶位點(diǎn)XhoI和BamHI之間，構(gòu)建成真核表達(dá)載體。
圖3中Ampr為芐青霉素抗性基因；flori為DNA單鏈復(fù)制起點(diǎn)；Synthetic poly(A)signal/transcriptional pause site為多聚腺苷酸合成信號/轉(zhuǎn)錄終止點(diǎn)，此序列可降低本底；KpnI.....HindIII為多限制性內(nèi)切酶位點(diǎn)序列，在此插入待檢測的DNA序列；Luc+為熒光素酶基因，是一種報告基因；SV40 late poly(A)signal為SV40病毒晚期轉(zhuǎn)錄單元的多聚腺苷酸合成信號；SalI，BamHI，HpaI，XbaI，NarI，NcoI分別是不同的限制性核酸內(nèi)切酶的位點(diǎn)，并表明其位點(diǎn)的序列位置。
圖4中SV40 Enhancer為SV40增強(qiáng)子序列；Ampr為芐青霉素抗性基因；flori為DNA單鏈復(fù)制起點(diǎn)；Synthetic poly(A)signal/transcriptional pause site為多聚腺苷酸合成信號/轉(zhuǎn)錄終止點(diǎn)，此序列可降低本底；KpnI.....HindIII為多限制性內(nèi)切酶位點(diǎn)序列，在此插入待檢測的DNA序列；Luc+為熒光素酶基因，是一種報告基因；SV40 latepoly(A)signal為SV40病毒晚期轉(zhuǎn)錄單元的多聚腺苷酸合成信號；SalI，BamHI，HpaI，XbaI，NarI，NcoI分別是不同的限制性核酸內(nèi)切酶的位點(diǎn)，并表明其位點(diǎn)的序列位置。
具體實施例方式
實施例1以Clontech公司的商售質(zhì)粒pcDNA3.1(-)為例，結(jié)合圖2，對本發(fā)明關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體作進(jìn)一步描述從關(guān)中奶山羊酪蛋白基因中提取基因組DNA作為模板，設(shè)計合成引物，用序列高保的DNA聚合酶進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)(簡稱PCR)擴(kuò)增，獲得的PCR產(chǎn)物，再經(jīng)過拼接、克隆和測定序列而得到的關(guān)中奶山羊β-酪蛋白基因-4359-+2106bp啟動區(qū)域序列(全長6465bp)；將全長6465bp的序列插入Clontech公司的商售質(zhì)粒pcDNA3.1(-)中的外源DNA限制性內(nèi)切酶位點(diǎn)XhoI和BamHI之間，得到本發(fā)明所需要的一種關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體；Clontech公司的商售質(zhì)粒pcDNA3.1(-)質(zhì)粒含有Pcmv CMV啟動子、BGHPA牛生長激素基因的多聚腺苷酸序列、Neomycin新霉素抗性基因、Ampicillin氨芐青霉素抗性基因、SV40pA病毒多聚腺苷酸序列，其DNA序列全長為5427bp，與所插入的關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列6465bp共同構(gòu)成全長為11838bp的序列，經(jīng)實際測試，結(jié)果與設(shè)計完全一致1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATGCTGCCTAGCC CTCTAGAGGG CTAGGGGATA CCACGTGAGA GTCATGTTAG ACGAGACTAC61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCGGGCGTATCAA TTCGGTCATA GACGAGGGAC GAACACACAA CCTCCAGCGA CTCATCACGC121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGCGCTCGTTTTA AATTCGATGT TGTTCCGTTC CGAACTGGCT GTTAACGTAC TTCTTAGACG181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATTAATCCCAATC CGCAAAACGC GACGAAGCGC TACATGCCCG GTCTATATGC GCAACTGTAA241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATACTAATAACTG ATCAATAATT ATCATTAGTT AATGCCCCAG TAATCAAGTA TCGGGTATAT301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACCACCTCAAGGC GCAATGTATT GAATGCCATT TACCGGGCGG ACCGACTGGC GGGTTGCTGG361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCCGGGCGGGTAA CTGCAGTTAT TACTGCATAC AAGGGTATCA TTGCGGTTAT CCCTGAAAGG421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGTTAACTGCAGT TACCCACCTC ATAAATGCCA TTTGACGGGT GAACCGTCAT GTAGTTCACA481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT
TAGTATACGG TTCATGCGGG GGATAACTGC AGTTACTGCC ATTTACCGGG CGGACCGTAA541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCATACGGGTCAT GTACTGGAAT ACCCTGAAAG GATGAACCGT CATGTAGATG CATAATCAGT601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTGAGCGATAATG GTACCACTAC GCCAAAACCG TCATGTAGTT ACCCGCACCT ATCGCCAAAC661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACCTGAGTGCCCC TAAAGGTTCA GAGGTGGGGT AACTGCAGTT ACCCTCAAAC AAAACCGTGG721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCGTTTTAGTTGC CCTGAAAGGT TTTACAGCAT TGTTGAGGCG GGGTAACTGC GTTTACCCGC781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCACATCCGCACA TGCCACCCTC CAGATATATT CGTCTCGAGA GACCGATTGA TCTCTTGGGT841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGCGACGAATGAC CGAATAGCTT TAATTATGCT GAGTGATATC CCTCTGGGTT CGACCGATCG901 GTTTAAACGG GCCCTCTAGA CAGATGATTT TGCAACCCCC TGCCTCAGGA GACACTGGGACAAATTTGCC CGGGAGATCT GTCTACTAAA ACGTTGGGGG ACGGAGTCCT CTGTGACCCT961 AATTTCCTGA GACATTTTTG ATTCCAAAAG CTGTGCAGTT GGTGCTTCTA CCATCTTCGTTTAAAGGACT CTGTAAAAAC TAAGGTTTTC GACACGTCAA CCACGAAGAT GGTAGAAGCA1021 GGTAGAGGTC AAGGATGCTG CTAAACATTC TACAACACAT TAAGAAAACC CCCACAACAACCATCTCCAG TTCCTACGAC GATTTGTAAG ATGTTGTGTA ATTCTTTTGG GGGTGTTGTT1081 AGAATTCTTC CGCCAAAAAT ATCAATAATA TGAAGGTTGA AAAATACTGG TCTAGCATGTTCTTAAGAAG GCGGTTTTTA TAGTTATTAT ACTTCCAACT TTTTATGACC AGATCGTACA1141 AGTATGTGCT CAATAGCAAG GAGAGAAAAG AAAGCCTTCC TCACTGATTA ATGCAAAGAATCATACACGA GTTATCGTTC CTCTCTTTTC TTTCGGAAGG AGTGACTAAT TACGTTTCTT1201 ATAGAGGAAA ACAATAGAAT GGGAAAGACT AGAGAGCTCT TCAAGCAAAT TAGAGATATCTATCTCCTTT TGTTATCTTA CCCTTTCTGA TCTCTCGAGA AGTTCGTTTA ATCTCTATAG1261 AAGGGAACAT TTCACGCAAA GATGGGCACA ATAAAGGACA GAAATTTTAT GGAGGAGTTGTTCCCTTGTA AAGTGCGTTT CTACCCGTGT TATTTCCTGT CTTTAAAATA CCTCCTCAAC1321 CTGATGGAGA GGGAGGCCTG GCGTGCTGCG ATTCCTGGGG TCGCAAAGAG TCGGACACAAGACTACCTCT CCCTCCGGAC CGCACGACGC TAAGGACCCC AGCGTTTCTC AGCCTGTGTT1381 CTGAGCGACT GAATTGAACT GAACTGAACT GGACAAAGCA GAAGATATTA AGAAGAGGTGGACTCGCTGA CTTAACTTGA CTTGACTTGA CCTGTTTCGT CTTCTATAAT TCTTCTCCAC1441 GTAAGAATAC ACAGAAGAAC AATATAAAAA AGATCTTCAT GACCCAGATA ACCACGATGACATTCTTATG TGTCTTCTTG TTATATTTTT TCTAGAAGTA CTGGGTCTAT TGGTGCTACT1501 TGTGATCACT CACCTAGAGC CAGACACCCT GGAATGCAAA GTCAAACGGC CTTAGAAAGCACACTAGTGA GTGGATCTCG GTCTGTGGGA CCTTACGTTT CAGTTTGCCG GAATCTTTCG1561 CTCACTATGA ACAAAGCTAG TGGAGGTAAT GGAATTCCAG TTGAGCTATT TCAAATCTTAGAGTGATACT TGTTTCGATC ACCTCCATTA CCTTAAGGTC AACTCGATAA AGTTTAGAAT1621 AAAGGTGATG CTGTGAAAGT GCTGCACTCA ATATGTCAGC AAATTTGGAA AACTCAGCAGTTTCCACTAC GACACTTTCA CGACGTGAGT TATACAGTCG TTTAAACCTT TTGAGTCGTC1681 TGGCCACAGG ACTGCCACAA TCCCAAAGAA AAGCAATGAC AAAGAATGTT CAAACACCCAACCGGTGTCC TGACGGTGTT AGGGTTTCTT TTCGTTACTG TTTCTTACAA GTTTGTGGGT1741 CATGATTGCA CTCATCTCAC ATGCTAGCAA AATAACTCTC AAAATTCTCC AAGCCAGGCTGTACTAACGT GAGTAGAGTG TACGATCGTT TTATTGAGAG TTTTAAGAGG TTCGGTCCGA1801 CCAACAGTAC GTGGACCATG AACTTCCAGA TGTTCAAGCT GGATTTAGAA AAGGCAGAGG
GGTTGTCATG CACCTGGTAC TTGAAGGTCT ACAAGTTCGA CCTAAATCTT TTCCGTCTCC1861 AACCAGAGAT CAAATTGCCA ACATCCATTG GATCATCAAA AAAGCACGAG AGTTCCAGAATTGGTCTCTA GTTTAACGGT TGTAGGTAAC CTAGTAGTTT TTTCGTGCTC TCAAGGTCTT1921 AAACATCTGC TTTATTGACT ACGCTAAAGC CTTTGATTGT GTGGATCACA ATAAACTGTGTTTGTAGACG AAATAACTGA TGCGATTTCG GAAACTAACA CACCTAGTGT TATTTGACAC1981 GAAAATTCTT CAAGAGATGG GAATACCAGA CCACTTTACC TGCCTCCTGA GAAATCTGTACTTTTAAGAA GTTCTCTACC CTTATGGTCT GGTGAAATGG ACGGAGGACT CTTTAGACAT2041 TACAGGTCCA GAAGCAGCAG TTAGAACTGG ACATGGAACA ACAGACTGGT TCCAAACTGCATGTCCAGGT CTTCGTCGTC AATCTTGACC TGTACCTTGT TGTCTGACCA AGGTTTGACG2101 GAAAGGGGTA CATCAAGGAA TATTCATTGG AAGGATTGAT GCTGAAGCTG AAACTCCTATCTTTCCCCAT GTAGTTCCTT ATAAGTAACC TTCCTAACTA CGACTTCGAC TTTGAGGATA2161 ACTTTGGCCA CCTAATGTGA AGATCTGACT CATTGGAAAA GACTCCAATG CTGGGAAAGATGAAACCGGT GGATTACACT TCTAGACTGA GTAACCTTTT CTGAGGTTAC GACCCTTTCT2221 TTGAAGGCAG GAGAAGAGGA TGACAGAGGA TGAGATGGTT GGATGGGATC ACTGACTCAAAACTTCCGTC CTCTTCTCCT ACTGTCTCCT ACTCTACCAA CCTACCCTAG TGACTGAGTT2281 TGGACATGAG TTTGAGTAAG CTCCAGGGGT TGGTGGTGGA CAGGAAAGCC TGGCGTGCTGACCTGTACTC AAACTCATTC GAGGTCCCCA ACCACCACCT GTCCTTTCGG ACCGCACGAC2341 CAGTCCACAA GGTCACAAAG ATTCGGACAT GACTGAGTGA CTGAACTGAT ACTGATGTGCGTCAGGTGTT CCAGTGTTTC TAAGCCTGTA CTGACTCACT GACTTGACTA TGACTACACG2401 TCAACAAATG TATCTTGAAC TTGTGTGAAG TTCTATGGTC ACATGTAAAG GAAGAATAATAGTTGTTTAC ATAGAACTTG AACACACTTC AAGATACCAG TGTACATTTC CTTCTTATTA2461 CAGGATTAGC TGTGTGTCTT AGGAATCAGG GTTCTGAGTT TTATGTGTTC ATAGTATCTGGTCCTAATCG ACACACAGAA TCCTTAGTCC CAAGACTCAA AATACACAAG TATCATAGAC2521 CTGGTTCACA AAACATTTTT CTTATTCTCT GGTTCTTGAT TTACTTTATA AAGTAATCTTGACCAAGTGT TTTGTAAAAA GAATAAGAGA CCAAGAACTA AATGAAATAT TTCATTAGAA2581 AATAGTTATA CTTCACATAG ATACGAAATT ATTATATTTG GATAATCTCA TGGAAAGGATTTATCAATAT GAAGTGTATC TATGCTTTAA TAATATAAAC CTATTAGAGT ACCTTTCCTA2641 TAAATACTCC ATCTATTACG AGTAATGCTG AACTATCTAC TCCTACCTAA TAATTTGTCAATTTATGAGG TAGATAATGC TCATTACGAC TTGATAGATG AGGATGGATT ATTAAACAGT2701 GAATTCACTA ATTCTGTGTT ATATTGTTTC TAAATCTGAA TCATTATATG AATCCTCAGTCTTAAGTGAT TAAGACACAA TATAACAAAG ATTTAGACTT AGTAATATAC TTAGGAGTCA2761 ATTTTGTTTT CCTTCCTCTA TATTTTGGAA TTTATTAAAC AGTGCTTCAA ATAATTTTTATAAAACAAAA GGAAGGAGAT ATAAAACCTT AAATAATTTG TCACGAAGTT TATTAAAAAT2821 GGAAACTGAA GTTTTTAGTA ACAGCTCTAT CTCTAAATAG CTTTAGTATC TTGAAAAAGTCCTTTGACTT CAAAAATCAT TGTCGAGATA GAGATTTATC GAAATCATAG AACTTTTTCA2881 AATACAAATT CTCACATCCT TAATTTCCTC TTCTCTAAAA TATCTTTAAA ATATTCTATGTTATGTTTAA GAGTGTAGGA ATTAAAGGAG AAGAGATTTT ATAGAAATTT TATAAGATAC2941 AATGATATCT CTTAATATTT ATTTTTTTGG CAATCCAACA CAGCTTATGG GATCTTAGTTTTACTATAGA GAATTATAAA TAAAAAAACC GTTAGGTTGT GTCGAATACC CTAGAATCAA3001 CCCCAGTGAG GGATTATATC CATGCCAACT GCAGTGAAAG TACAAAATCC TAAACTGGACGGGGTCACTC CCTAATATAG GTACGGTTGA CGTCACTTTC ATGTTTTAGG ATTTGACCTG3061 TCACCAGGGA TTTCCCAATA TCTCCTCTAG TTCTTATTTC TGAATATTTT TGGTCCCTTTAGTGGTCCCT AAAGGGTTAT AGAGGAGATC AAGAATAAAG ACTTATAAAA ACCAGGGAAA3121 ATTGTACTCT TCATCCAACT TTTCTATTGA TTTCTTTCTT GAGGTTATTA TTTACTTGGT
TAACATGAGA AGTAGGTTGA AAAGATAACT AAAGAAAGAA CTCCAATAAT AAATGAACCA3181 TTCAGTTAGA AATATATGCA AATCTCAGGA CTGCATATTT CAGATTCATT GGCCAATATGAAGTCAATCT TTATATACGT TTAGAGTCCT GACGTATAAA GTCTAAGTAA CCGGTTATAC3241 GGAAAAAACC TTTGGCTGAA CAAATCATGC TTATAAAAAA TAGTACTAGA GCATCCTACTCCTTTTTTGG AAACCGACTT GTTTAGTACG AATATTTTTT ATCATGATCT CGTAGGATGA3301 TTGACTATAT CTTGCTCCTC ATTCAGGGTT ATCTAATACA ATTTCCCCAC ATGAAATTCTAACTGATATA GAACGAGGAG TAAGTCCCAA TAGATTATGT TAAAGGGGTG TACTTTAAGA3361 TTTGCATTAT AAAAATGGAA GCTCTTAGGT AACATTGCAA AAATTCGAGT TGCTCATATGAAACGTAATA TTTTTACCTT CGAGAATCCA TTGTAACGTT TTTAAGCTCA ACGAGTATAC3421 GCACTTTGCT TCTTACTGGT CATTGTGTTC TGAGGCTTAC CTGGACAGGT GGTACCTGATCGTGAAACGA AGAATGACCA GTAACACAAG ACTCCGAATG GACCTGTCCA CCATGGACTA3481 GTCATCTTAA ATTGCTGGCT TTTTGATTTT CCATTGGACA AGCTTCTTTC TTTAGTATATCAGTAGAATT TAACGACCGA AAAACTAAAA GGTAACCTGT TCGAAGAAAG AAATCATATA3541 TGTTAAGGAT TTCCTTGATC AAGATTTTAC CTACTTTTCT GGTCCAATTG GTGAGAGACAACAATTCCTA AAGGAACTAG TTCTAAAATG GATGAAAAGA CCAGGTTAAC CACTCTCTGT3601 GTCATAAGGA AATGCTGTGT TTATTGCACA ATATGTAAAG CATCTTCCTG AGAAAATAAACAGTATTCCT TTACGACACA AATAACGTGT TATACATTTC GTAGAAGGAC TCTTTTATTT3661 AGGGAAATGT TGAATGGGAA GGATATGCTT TCTTTTGTAT TCCTTTTCTG AGAAATCAAATCCCTTTACA ACTTACCCTT CCTATACGAA AGAAAACATA AGGAAAAGAC TCTTTAGTTT3721 CTTTTTCACC TGTGGCCTTG GCCACCAAAA GCTAACAAAT AAAGGCATAT GAAGTAGCCAGAAAAAGTGG ACACCGGAAC CGGTGGTTTT CGATTGTTTA TTTCCGTATA CTTCATCGGT3781 AGGCCTTTTC TAGTTATATC TATAACACTG AGTTCATTTC ATCATTTATT TTCCTGACTTTCCGGAAAAG ATCAATATAG ATATTGTGAC TCAAGTAAAG TAGTAAATAA AAGGACTGAA3841 CCTCCTGGGT CCATATGAGC AGTCTTAGAA TGAATATTAG CTGAATAATC CAAATACATAGGAGGACCCA GGTATACTCG TCAGAATCTT ACTTATAATC GACTTATTAG GTTTATGTAT3901 GTAGATGTTG ATTTGGGTTT TCTAAGCAAT CCAAGACTTG TATGACAGTA AGATGTATTACATCTACAAC TAAACCCAAA AGATTCGTTA GGTTCTGAAC ATACTGTCAT TCTACATAAT3961 CCATCCAACA CACATCTCAG CATGATATAA ATGCAAGGTA TATTGTGAAG AAAAATTTTTGGTAGGTTGT GTGTAGAGTC GTACTATATT TACGTTCCAT ATAACACTTC TTTTTAAAAA4021 AATTATGTCA AAGTGCTTAC TTTAGAAGGT CATCTATCTG TCCCAAAGCT GTGAATATATTTAATACAGT TTCACGAATG AAATCTTCCA GTAGATAGAC AGGGTTTCGA CACTTATATA4081 ATATTGAAGG TAATGAATAG ATGAAGCTAA CCTTGTAAAA ATGAGTAGTG TGAAATACAATATAACTTCC ATTACTTATC TACTTCGATT GGAACATTTT TACTCATCAC ACTTTATGTT4141 CTACAATTAT GAACATCTGT CACTAAAGAG GCAAAGAAAC TTGAAGATTG CTTTTGCAAAGATGTTAATA CTTGTAGACA GTGATTTCTC CGTTTCTTTG AACTTCTAAC GAAAACGTTT4201 TGGGCTCCTA TTAATAAAAA GTACTTTTGA GGTCTGGCTC AGACTCTATT GTAGTACTTAACCCGAGGAT AATTATTTTT CATGAAAACT CCAGACCGAG TCTGAGATAA CATCATGAAT4261 GGGTAAGACC CTCCTCCTGT ATGGGCTTTC ATTTTCTTTC TTGCTTCCCT CATTTGCCCTCCCATTCTGG GAGGAGGACA TACCCGAAAG TAAAAGAAAG AACGAAGGGA GTAAACGGGA4321 TCCATGAATA CTAGCTGATA AACATTGACT ATAAAAGATA TGAGGCCAAA CTTGAGCTGTAGGTACTTAT GATCGACTAT TTGTAACTGA TATTTTCTAT ACTCCGGTTT GAACTCGACA4381 CCCATTTTAA TAAATCTGTA TAAATAATAT TTGTTCTACA AAAGTATTAT CTAAATAAATGGGTAAAATT ATTTAGACAT ATTTATTATA AACAAGATGT TTTCATAATA GATTTATTTA4441 GTTACTTTCT GTCTTAAAAT CCCTCAACAA ATCCCCACTA TCTAGAGAAT AAGATTGACA
CAATGAAAGA CAGAATTTTA GGGAGTTGTT TAGGGGTGAT AGATCTCTTA TTCTAACTGT4501 TTCCCTGGAA TCACAGCATG CTTTGTCTGC CATTATCTGA CCCCTTTCTC TTTCTCTCTTAAGGGACCTT AGTGTCGTAC GAAACAGACG GTAATAGACT GGGGAAAGAG AAAGAGAGAA4561 CTCACCTCCA TCTACTCCTT TTTCCTTGCA ATTCATGACC CAGATTCACT GTTTGATTTGGAGTGGAGGT AGATGAGGAA AAAGGAACGT TAAGTACTGG GTCTAAGTGA CAAACTAAAC4621 GCTTGCATGT GTGTGTGCTG AGTTGCGTCT GACTGTTATC AACCCCATGA ATGATAGTCCCGAACGTACA CACACACGAC TCAACGCAGA CTGACAATAG TTGGGGTACT TACTATCAGG4681 ACCAGGCTCT ACTGTCCATG AAATTTTCCA GTCAAGAATA CTGGAGTGGA TTGCATTTCCTGGTCCGAGA TGACAGGTAC TTTAAAAGGT CAGTTCTTAT GACCTCACCT AACGTAAAGG4741 TACTCCATTT GATTAATTTA GTGACTTTTA AATTTCTTTT TCCATATTCG GGAGCCTATTATGAGGTAAA CTAATTAAAT CACTGAAAAT TTAAAGAAAA AGGTATAAGC CCTCGGATAA4801 CTTCCTTTTT AGTCTATACT CTCTTCACTC TTCAGGTCTA AGGTATCATC GTGTGCTTGTGAAGGAAAAA TCAGATATGA GAGAAGTGAG AAGTCCAGAT TCCATAGTAG CACACGAACA4861 TAGCTTGTTA CTTTCTCCAT TATAGCTTAA GCACTAACAA CTGTTCAGGT TGGCATGAAAATCGAACAAT GAAAGAGGTA ATATCGAATT CGTGATTGTT GACAAGTCCA ACCGTACTTT4921 TTGTGTTCTT TGTGTGGCCT GTATATTTCT GTTGTGTATT AGAATTTACC CCAAGATCTCAACACAAGAA ACACACCGGA CATATAAAGA CAACACATAA TCTTAAATGG GGTTCTAGAG4981 AAAGACCCAC TGAATACTAA AGAGACCTCA TTGTGGTTAC AATAATTTGG GGACTGGGCCTTTCTGGGTG ACTTATGATT TCTCTGGAGT AACACCAATG TTATTAAACC CCTGACCCGG5041 AAAACTACCG TGCATCCCAG CCAAGATCTG TAGCTACTGG ACAATTTCAT TTCCTTTATCTTTTGATGGC ACGTAGGGTC GGTTCTAGAC ATCGATGACC TGTTAAAGTA AAGGAAATAG5101 AGATTGTGAG TTATTCCTGT TAAAATGCTC CCCAGAATTT CTGGGGACAG AAAAATAGGATCTAACACTC AATAAGGACA ATTTTACGAG GGGTCTTAAA GACCCCTGTC TTTTTATCCT5161 AGAATTCATT TCCTAATCAT GCAGATTTCT AGGAATTCAA ATCCACTGTT GGTTTTATTTTCTTAAGTAA AGGATTAGTA CGTCTAAAGA TCCTTAAGTT TAGGTGACAA CCAAAATAAA5221 CAAACCACAA AATTAGCATG CCATTAAATA CTATATATAA ACAGCCACTA AATCAGATCAGTTTGGTGTT TTAATCGTAC GGTAATTTAT GATATATATT TGTCGGTGAT TTAGTCTAGT5281 TTATCCATTC AGCTTCTCCT TCACTTCTTC TCCTCTACTT TGGAAAAAAG GTAAGAATCTAATAGGTAAG TCGAAGAGGA AGTGAAGAAG AGGAGATGAA ACCTTTTTTC CATTCTTAGA5341 CAGATATAAT TTCAGGTGTA TCTGCTACTC ATCTTTATTT TGGACTAGGT TAAAATGTAGGTCTATATTA AAGTCCACAT AGACGATGAG TAGAAATAAA ACCTGATCCA ATTTTACATC5401 AAAGAACATA ATTGCTTAAA ATAGATCTTA AAAATAAGGG TGTTTAAGAT AAGGTTTACATTTCTTGTAT TAACGAATTT TATCTAGAAT TTTTATTCCC ACAAATTCTA TTCCAAATGT5461 CTATTTTCAG CAGATATGTT AAAAAATAGA AGTGACTATA AAGACTTGAT AAAAATTATAGATAAAAGTC GTCTATACAA TTTTTTATCT TCACTGATAT TTCTGAACTA TTTTTAATAT5521 GTGACTGCAA ATGTTTTAGG AATATAATAA GATATAATAA CGGTGGTTGC TATTTTCTTTCACTGACGTT TACAAAATCC TTATATTATT CTATATTATT GCCACCAACG ATAAAAGAAA5581 AGCACAAGAC TAGTTAACAG GCTGTATTAA AAGATCTTTT CTTGAATTAA ATATTTTCAATCGTGTTCTG ATCAATTGTC CGACATAATT TTCTAGAAAA GAACTTAATT TATAAAAGTT5641 TTTGATTAAA CCTACCTCAG CCATAAAGGC AAGCACATTT CATTTATACT ATGGGGATTTAAACTAATTT GGATGGAGTC GGTATTTCCG TTCGTGTAAA GTAAATATGA TACCCCTAAA5701 GAATAATTAT TACTGAAGAA GCTCTACCAA CAAAAAGTTT ATAGAGCTAT CATATTTAGTCTTATTAATA ATGACTTCTT CGAGATGGTT GTTTTTCAAA TATCTCGATA GTATAAATCA5761 CAAGAGATAA AGAGGGTTGT TAGGATATAT ATGCTATTTG AAAGGTATTT ATAAAAGAAG
GTTCTCTATT TCTCCCAACA ATCCTATATA TACGATAAAC TTTCCATAAA TATTTTCTTC5821 AGTATATTTA TCAAAATTTC TCAGAACATC CAAATTTCAA GTTTATCATT TATCTTACAATCATATAAAT AGTTTTAAAG AGTCTTGTAG GTTTAAAGTT CAAATAGTAA ATAGAATGTT5881 TATTTCAAAA ATATTAAAAT AGATACTGAA ATACAGAAGT AAATTAAAGA GAAAGTATTTATAAAGTTTT TATAATTTTA TCTATGACTT TATGTCTTCA TTTAATTTCT CTTTCATAAA5941 TACTTGGTAA AAAAATTCTA GGTTGGACAG AGAGTGCCAG GAAACAAAAA CAATGAAAAAATGAACCATT TTTTTAAGAT CCAACCTGTC TCTCACGGTC CTTTGTTTTT GTTACTTTTT6001 TGTGACCTGA CAGGAATTAT AGCTCAAAGT ATAGTAGTAA GTAATGAAAT GGCTTAAAAAACACTGGACT GTCCTTAATA TCGAGTTTCA TATCATCATT CATTACTTTA CCGAATTTTT6061 TTGGTATATA AAATGCTAGT TATAAAATAA ACAAAATGCA ATAATATCCT CCCTACATGTAACCATATAT TTTACGATCA ATATTTTATT TGTTTTACGT TATTATAGGA GGGATGTACA6121 AATGAATTCT AGGTATTATG CTCTTTTTGG AAGTCTTGAC AATAAAAATT TTTTTAGAAGTTACTTAAGA TCCATAATAC GAGAAAAACC TTCAGAACTG TTATTTTTAA AAAAATCTTC6181 TTTATAGGCA TCTTGAATAA AGTGAAACAA ATTAAGAATT AGTATCCATG AGAAAAATATAAATATCCGT AGAACTTATT TCACTTTGTT TAATTCTTAA TCATAGGTAC TCTTTTTATA6241 AGAACAATTT TCCTAATTTA GTTTGAAAAT CTGGGATTGA AGATGTGTGT CAAGAGATGTTCTTGTTAAA AGGATTAAAT CAAACTTTTA GACCCTAACT TCTACACACA GTTCTCTACA6301 TGGTGGCAAG AACATTTTTT TTTCAAGAAC TTATAAAAAT GCAACAAAAC AAACCATTTAACCACCGTTC TTGTAAAAAA AAAGTTCTTG AATATTTTTA CGTTGTTTTG TTTGGTAAAT6361 ATACATTTTG GTCAAAATCA ATAATGTATT TTATTTTATG CTCCAAGGAG CATAAAATTGTATGTAAAAC CAGTTTTAGT TATTACATAA AATAAAATAC GAGGTTCCTC GTATTTTAAC6421 GGGACTGGGC AAGAGAAACT GACACCCTGG TAAATTACCA AGAGATAAGT ACACAGTTCTCCCTGACCCG TTCTCTTTGA CTGTGGGACC ATTTAATGGT TCTCTATTCA TGTGTCAAGA6481 ATGTAGAGAA AATAAGCATA GTGTATGATC TCTAAAATTA TGTGAGACAA AGGAGAGATGTACATCTCTT TTATTCGTAT CACATACTAG AGATTTTAAT ACACTCTGTT TCCTCTCTAC6541 ACATTAGGCA TGTGGGGATG AAGACTGAGT AGAGAAGAAA CAATCTAATC AGTCCAAGAATGTAATCCGT ACACCCCTAC TTCTGACTCA TCTCTTCTTT GTTAGATTAG TCAGGTTCTT6601 AACATCTCGA TCAGTGGAAC AAATAGAAGA AATGCTAAAA TGAAACAGAA GTCTTACTGGTTGTAGAGCT AGTCACCTTG TTTATCTTCT TTACGATTTT ACTTTGTCTT CAGAATGACC6661 AAATAAAAGA TATGCATAAG ACAAAAATTC ATGAAAATCA CTTAGTTTAG CAGAGAAAAGTTTATTTTCT ATACGTATTC TGTTTTTAAG TACTTTTAGT GAATCAAATC GTCTCTTTTC6721 ATAAAAATAA AGTATGACCT TCTTCATATA CATTGTTTGA TCATATGCAC CTCAATAAAATATTTTTATT TCATACTGGA AGAAGTATAT GTAACAAACT AGTATACGTG GAGTTATTTT6781 CTGAGTCTCC AACAGAAATG AAACATTAAT ATTTTGTTCA CTGCTCTAAT CCCAGAATCTGACTCAGAGG TTGTCTTTAC TTTGTAATTA TAAAACAAGT GACGAGATTA GGGTCTTAGA6841 AAGCGATATC TGGCAATAAA AATAATAAAT ATATATTTTT TAATAAATGA ATCAACCACTTTCGCTATAG ACCGTTATTT TTATTATTTA TATATAAAAA ATTATTTACT TAGTTGGTGA6901 TAATTTTTCT GTAAATATCT GTAACTTCTC TTCTGTCTTT CCAAAAACAC TCATAAGTACATTAAAAAGA CATTTATAGA CATTGAAGAG AAGACAGAAA GGTTTTTGTG AGTATTCATG6961 TGTGAATGAG ATGAAAAAGA GTGAAGTAGG ATATAGGCTG TTAGCAGAAA ACATCTGAATACACTTACTC TACTTTTTCT CACTTCATCC TATATCCGAC AATCGTCTTT TGTAGACTTA7021 GGCTGGCAGT GAAACATTAA CTTGAAATGT AAGATTAATG AGTAATAGTA AATTTTAACCCCGACCGTCA CTTTGTAATT GAACTTTACA TTCTAATTAC TCATTATCAT TTAAAATTGG7081 TTGGCCATAT GATAAAATGT TCATTAATAT TTTTCTAGAA TACAGGGCTT TTTGTTTTTG
AACCGGTATA CTATTTTACA AGTAATTATA AAAAGATCTT ATGTCCCGAA AAACAAAAAC7141 CCATGAGGTT TGCAGGATCT TGGTTCCCTG ACCAGGGATC AAACCTGCAC TCCCCTGGAAGGTACTCCAA ACGTCCTAGA ACCAAGGGAC TGGTCCCTAG TTTGGACGTG AGGGGACCTT7201 GCATGGAGTC TTGGACATTT GTATTATACA CTATCTTTGG TTCCTTTTAA AGGGAAGTAACGTACCTCAG AACCTGTAAA CATAATATGT GATAGAAACC AAGGAAAATT TCCCTTCATT7261 TTTTACTTAA ATAAGAAAAT AGATTGACAA GTAATACGCT GTTTCCTCAT CTTCCCATTCAAAATGAATT TATTCTTTTA TCTAACTGTT CATTATGCGA CAAAGGAGTA GAAGGGTAAG7321 ACAGGAATCG AGAGCCATGA AGGTCCTCAT CCTTGCCTGT CTGGTGGCTC TGGCCATTGCTGTCCTTAGC TCTCGGTACT TCCAGGAGTA GGAACGGACA GACCACCGAG ACCGGTAACG7381 GATCGCGGAT CCGAGCTCGG TACCAAGCTT AAGTTTAAAC CCGCTGATCA GCCTCGACTGCTAGCGCCTA GGCTCGAGCC ATGGTTCGAA TTCAAATTTG GGCGACTAGT CGGAGCTGAC7441 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGGACGGAAGATC AACGGTCGGT AGACAACAAA CGGGGAGGGG GCACGGAAGG AACTGGGACC7501 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGATTCCACGGTG AGGGTGACAG GAAAGGATTA TTTTACTCCT TTAACGTAGC GTAACAGACT7561 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGGCATCCACAGT AAGATAAGAC CCCCCACCCC ACCCCGTCCT GTCGTTCCCC CTCCTAACCC7621 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAATTCTGTTATC GTCCGTACGA CCCCTACGCC ACCCGAGATA CCGAAGACTC CGCCTTTCTT7681 CCAGCTGGGG CTCTAGGGGG TATCCCCACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGGGGTCGACCCC GAGATCCCCC ATAGGGGTGC GCGGGACATC GCCGCGTAAT TCGCGCCGCC7741 GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTTCACACCACCA ATGCGCGTCG CACTGGCGAT GTGAACGGTC GCGGGATCGC GGGCGAGGAA7801 TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATCAGCGAAAGAA GGGAAGGAAA GAGCGGTGCA AGCGGCCGAA AGGGGCAGTT CGAGATTTAG7861 GGGGGCTCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTGCCCCCGAGGG AAATCCCAAG GCTAAATCAC GAAATGCCGT GGAGCTGGGG TTTTTTGAAC7921 ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGATAATCCCACT ACCAAGTGCA TCACCCGGTA GCGGGACTAT CTGCCAAAAA GCGGGAAACT7981 CGTTGGAGTC CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACCGCAACCTCAG GTGCAAGAAA TTATCACCTG AGAACAAGGT TTGACCTTGT TGTGAGTTGG8041 CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC TATTGGTTAAGATAGAGCCA GATAAGAAAA CTAAATATTC CCTAAAACGG CTAAAGCCGG ATAACCAATT8101 AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG TGTGTCAGTTTTTTACTCGA CTAAATTGTT TTTAAATTGC GCTTAATTAA GACACCTTAC ACACAGTCAA8161 AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAATCCCACACCT TTCAGGGGTC CGAGGGGTCG TCCGTCTTCA TACGTTTCGT ACGTAGAGTT8221 TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAGAATCAGTCGT TGGTCCACAC CTTTCAGGGG TCCGAGGGGT CGTCCGTCTT CATACGTTTC8281 CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCTGTACGTAGAG TTAATCAGTC GTTGGTATCA GGGCGGGGAT TGAGGCGGGT AGGGCGGGGA8341 AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT TTATTTATGCTTGAGGCGGG TCAAGGCGGG TAAGAGGCGG GGTACCGACT GATTAAAAAA AATAAATACG8401 AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG
TCTCCGGCTC CGGCGGAGAC GGAGACTCGA TAAGGTCTTC ATCACTCCTC CGAAAAAACC8461 AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA TCCATTTTCG GATCTGATCATCCGGATCCG AAAACGTTTT TCGAGGGCCC TCGAACATAT AGGTAAAAGC CTAGACTAGT8521 AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCCTCTCTGTCCT ACTCCTAGCA AAGCGTACTA ACTTGTTCTA CCTAACGTGC GTCCAAGAGG8581 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTCCCGGCGAACC CACCTCTCCG ATAAGCCGAT ACTGACCCGT GTTGTCTGTT AGCCGACGAG8641 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGAACTACGGCGG CACAAGGCCG ACAGTCGCGT CCCCGCGGGC CAAGAAAAAC AGTTCTGGCT8701 CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT GGCTGGCCACGGACAGGCCA CGGGACTTAC TTGACGTCCT GCTCCGTCGC GCCGATAGCA CCGACCGGTG8761 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCTCTGCCCGCAA GGAACGCGTC GACACGAGCT GCAACAGTGA CTTCGCCCTT CCCTGACCGA8821 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAACGATAACCCG CTTCACGGCC CCGTCCTAGA GGACAGTAGA GTGGAACGAG GACGGCTCTT8881 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCCTCATAGGTAG TACCGACTAC GTTACGCCGC CGACGTATGC GAACTAGGCC GATGGACGGG8941 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCTTAAGCTGGTG GTTCGCTTTG TAGCGTAGCT CGCTCGTGCA TGAGCCTACC TTCGGCCAGA9001 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGCACAGCTAGTC CTACTAGACC TGCTTCTCGT AGTCCCCGAG CGCGGTCGGC TTGACAAGCG9061 CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTGGTCCGAGTTC CGCGCGTACG GGCTGCCGCT CCTAGAGCAG CACTGGGTAC CGCTACGGAC9121 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCTGAACGGCTTA TAGTACCACC TTTTACCGGC GAAAAGACCT AAGTAGCTGA CACCGGCCGA9181 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCTCCCACACCGC CTGGCGATAG TCCTGTATCG CAACCGATGG GCACTATAAC GACTTCTCGA9241 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCAACCGCCGCTT ACCCGACTGG CGAAGGAGCA CGAAATGCCA TAGCGGCGAG GGCTAAGCGT9301 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAACGCGTAGCGG AAGATAGCGG AAGAACTGCT CAAGAAGACT CGCCCTGAGA CCCCAAGCTT9361 ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTCTACTGGCTGG TTCGCTGCGG GTTGGACGGT AGTGCTCTAA AGCTAAGGTG GCGGCGGAAG9421 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGCATACTTTCCA ACCCGAAGCC TTAGCAAAAG GCCCTGCGGC CGACCTACTA GGAGGTCGCG9481 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC TTATAATGGTCCCCTAGAGT ACGACCTCAA GAAGCGGGTG GGGTTGAACA AATAACGTCG AATATTACCA9541 TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCTATGTTTATTT CGTTATCGTA GTGTTTAAAG TGTTTATTTC GTAAAAAAAG TGACGTAAGA9601 AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCTTCAACACCAA ACAGGTTTGA GTAGTTACAT AGAATAGTAC AGACATATGG CAGCTGGAGA9661 AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTCTCGATCTCGA ACCGCATTAG TACCAGTATC GACAAAGGAC ACACTTTAAC AATAGGCGAG9721 ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA
TGTTAAGGTG TGTTGTATGC TCGGCCTTCG TATTTCACAT TTCGGACCCC ACGGATTACT9781 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTGCACTCGATTG AGTGTAATTA ACGCAACGCG AGTGACGGGC GAAAGGTCAG CCCTTTGGAC9841 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGGAGCACGGTCG ACGTAATTAC TTAGCCGGTT GCGCGCCCCT CTCCGCCAAA CGCATAACCC9901 CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCGGCGAGAAGGC GAAGGAGCGA GTGACTGAGC GACGCGAGCC AGCAAGCCGA CGCCGCTCGC9961 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGACATAGTCGAG TGAGTTTCCG CCATTATGCC AATAGGTGTC TTAGTCCCCT ATTGCGTCCT10021 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTGTTCTTGTACA CTCGTTTTCC GGTCGTTTTC CGGTCCTTGG CATTTTTCCG GCGCAACGAC10081 GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAGCGCAAAAAGG TATCCGAGGC GGGGGGACTG CTCGTAGTGT TTTTAGCTGC GAGTTCAGTC10141 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTCTCCACCGCTT TGGGCTGTCC TGATATTTCT ATGGTCCGCA AAGGGGGACC TTCGAGGGAG10201 GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCGCACGCGAGAG GACAAGGCTG GGACGGCGAA TGGCCTATGG ACAGGCGGAA AGAGGGAAGC10261 GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTTCCTTCGCACC GCGAAAGAGT ATCGAGTGCG ACATCCATAG AGTCAAGCCA CATCCAGCAA10321 CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCCGCGAGGTTCG ACCCGACACA CGTGCTTGGG GGGCAAGTCG GGCTGGCGAC GCGGAATAGG10381 GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCCCCATTGATAG CAGAACTCAG GTTGGGCCAT TCTGTGCTGA ATAGCGGTGA CCGTCGTCGG10441 ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGGTGACCATTGT CCTAATCGTC TCGCTCCATA CATCCGCCAC GATGTCTCAA GAACTTCACC10501 TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCAACCGGATTGA TGCCGATGTG ATCTTCTTGT CATAAACCAT AGACGCGAGA CGACTTCGGT10561 GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGCCAATGGAAGC CTTTTTCTCA ACCATCGAGA ACTAGGCCGT TTGTTTGGTG GCGACCATCG10621 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCTCCAAAAAAAC AAACGTTCGT CGTCTAATGC GCGTCTTTTT TTCCTAGAGT TCTTCTAGGA10681 TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTGAACTAGAAAA GATGCCCCAG ACTGCGAGTC ACCTTGCTTT TGAGTGCAAT TCCCTAAAAC10741 GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTTCAGTACTCTA ATAGTTTTTC CTAGAAGTGG ATCTAGGAAA ATTTAATTTT TACTTCAAAA10801 AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGTTTTAGTTAGA TTTCATATAT ACTCATTTGA ACCAGACTGT CAATGGTTAC GAATTAGTCA10861 GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTCCTCCGTGGAT AGAGTCGCTA GACAGATAAA GCAAGTAGGT ATCAACGGAC TGAGGGGCAG10921 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCGCACATCTATT GATGCTATGC CCTCCCGAAT GGTAGACCGG GGTCACGACG TTACTATGGC10981 CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCCGCTCTGGGTG CGAGTGGCCG AGGTCTAAAT AGTCGTTATT TGGTCGGTCG GCCTTCCCGG11041 GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG
CTCGCGTCTT CACCAGGACG TTGAAATAGG CGGAGGTAGG TCAGATAATT AACAACGGCC11101 GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACACTTCGATCTC ATTCATCAAG CGGTCAATTA TCAAACGCGT TGCAACAACG GTAACGATGT11161 GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGACCGTAGCACC ACAGTGCGAG CAGCAAACCA TACCGAAGTA AGTCGAGGCC AAGGGTTGCT11221 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCTAGTTCCGCTC AATGTACTAG GGGGTACAAC ACGTTTTTTC GCCAATCGAG GAAGCCAGGA11281 CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTGGGCTAGCAAC AGTCTTCATT CAACCGGCGT CACAATAGTG AGTACCAATA CCGTCGTGAC11341 CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCAGTATTAAGAG AATGACAGTA CGGTAGGCAT TCTACGAAAA GACACTGACC ACTCATGAGT11401 ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATATGGTTCAGTA AGACTCTTAT CACATACGCC GCTGGCTCAA CGAGAACGGG CCGCAGTTAT11461 CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCTGCCCTATTAT GGCGCGGTGT ATCGTCTTGA AATTTTCACG AGTAGTAACC TTTTGCAAGA11521 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACTAGCCCCGCTT TTGAGAGTTC CTAGAATGGC GACAACTCTA GGTCAAGCTA CATTGGGTGA11581 CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAAGCACGTGGGT TGACTAGAAG TCGTAGAAAA TGAAAGTGGT CGCAAAGACC CACTCGTTTT11641 ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTCTGTCCTTCCG TTTTACGGCG TTTTTTCCCT TATTCCCGCT GTGCCTTTAC AACTTATGAG11701 ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGATATGAGAAGG AAAAAGTTAT AATAACTTCG TAAATAGTCC CAATAACAGA GTACTCGCCT11761 TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGAATGTATAAAC TTACATAAAT CTTTTTATTT GTTTATCCCC AAGGCGCGTG TAAAGGGGCT11821 AAAGTGCCAC CTGACGTCTTTCACGGTG GACTGCAG實施例2以Promega公司的商售pGL3-Basic Vector質(zhì)粒為例，結(jié)合圖3，對本發(fā)明關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體作進(jìn)一步描述實施例3以Promega公司的商售pGL3-Enhancer Vector質(zhì)粒為例，結(jié)合圖4，對本發(fā)明關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體作進(jìn)一步描述從關(guān)中奶山羊酪蛋白基因中提取基因組DNA作為模板，設(shè)計合成引物，用序列高保真的DNA聚合酶進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)(簡稱PCR)擴(kuò)增，獲得的PCR產(chǎn)物，再經(jīng)過拼接、克隆和測定序列而得到的關(guān)中奶山羊β-酪蛋白基因-4359～+2106bp啟動區(qū)域序列(全長6465bp)；將全長6465bp的序列插入Promega公司的商售pGL3-EnhancerVector質(zhì)粒中KpnI和BglII多限制性內(nèi)切酶位點(diǎn)之間，得到本發(fā)明所需要的另一種關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體；Promega公司的商售pGL3-Basic Vector質(zhì)粒中含有Ampr芐青霉素抗性基因、Luc+為熒光素酶基因、SV40病毒晚期轉(zhuǎn)錄單元的多聚腺苷酸合成信號，SV40病毒增強(qiáng)子基因序列。
權(quán)利要求
1.關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體，具有商售質(zhì)粒載體和β-酪蛋白基因啟動子及其周圍的調(diào)控序列以及第一內(nèi)含子、第一外顯子和第二外顯子所構(gòu)成的β-酪蛋白基因啟動子區(qū)域，其特征在于(1)、所述的β-酪蛋白基因啟動子區(qū)域序列還包括在第二外顯子末端引入作為信號肽的限制性核酸內(nèi)切酶Sgf I位點(diǎn)序列；該啟動區(qū)域序列為-4359～+2106bp，全長6465bp；(2)、所述的β-酪蛋白基因啟動子是以西北楊凌特有的高產(chǎn)關(guān)中奶山羊的乳腺組織作為來源，從中提取基因組DNA作為模板，設(shè)計合成引物，用高保真的DNA聚合酶進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)(PCR)，獲得的PCR產(chǎn)物，經(jīng)過拼接、克隆和測定序列而得；(3)、所述的關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體是將含關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列插入商售的質(zhì)粒載體中后得到的關(guān)中奶山羊β-酪蛋白基因啟動子表達(dá)載體，其DNA序列全長是關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列全長6465bp與商售的質(zhì)粒載體序列全長之和。
2.根據(jù)權(quán)利要求1所述的關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體，其特征在于關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列全長6465bp如下所示1 AGATGATTTT GCAACCCCCT GCCTCAGGAG ACACTGGGAA ATTTCCTGAG ACATTTTTGATCTACTAAAA CGTTGGGGGA CGGAGTCCTC TGTGACCCTT TAAAGGACTC TGTAAAAACT61 TTCCAAAAGC TGTGCAGTTG GTGCTTCTAC CATCTTCGTG GTAGAGGTCA AGGATGCTGCAAGGTTTTCG ACACGTCAAC CACGAAGATG GTAGAAGCAC CATCTCCAGT TCCTACGACG121 TAAACATTCT ACAACACATT AAGAAAACCC CCACAACAAA GAATTCTTCC GCCAAAAATAATTTGTAAGA TGTTGTGTAA TTCTTTTGGG GGTGTTGTTT CTTAAGAAGG CGGTTTTTAT181 TCAATAATAT GAAGGTTGAA AAATACTGGT CTAGCATGTA GTATGTGCTC AATAGCAAGGAGTTATTATA CTTCCAACTT TTTATGACCA GATCGTACAT CATACACGAG TTATCGTTCC241 AGAGAAAAGA AAGCCTTCCT CACTGATTAA TGCAAAGAAA TAGAGGAAAA CAATAGAATGTCTCTTTTCT TTCGGAAGGA GTGACTAATT ACGTTTCTTT ATCTCCTTTT GTTATCTTAC301 GGAAAGACTA GAGAGCTCTT CAAGCAAATT AGAGATATCA AGGGAACATT TCACGCAAAGCCTTTCTGAT CTCTCGAGAA GTTCGTTTAA TCTCTATAGT TCCCTTGTAA AGTGCGTTTC361 ATGGGCACAA TAAAGGACAG AAATTTTATG GAGGAGTTGC TGATGGAGAG GGAGGCCTGGTACCCGTGTT ATTTCCTGTC TTTAAAATAC CTCCTCAACG ACTACCTCTC CCTCCGGACC421 CGTGCTGCGA TTCCTGGGGT CGCAAAGAGT CGGACACAAC TGAGCGACTG AATTGAACTGGCACGACGCT AAGGACCCCA GCGTTTCTCA GCCTGTGTTG ACTCGCTGAC TTAACTTGAC481 AACTGAACTG GACAAAGCAG AAGATATTAA GAAGAGGTGG TAAGAATACA CAGAAGAACATTGACTTGAC CTGTTTCGTC TTCTATAATT CTTCTCCACC ATTCTTATGT GTCTTCTTGT541 ATATAAAAAA GATCTTCATG ACCCAGATAA CCACGATGAT GTGATCACTC ACCTAGAGCCTATATTTTTT CTAGAAGTAC TGGGTCTATT GGTGCTACTA CACTAGTGAG TGGATCTCGG601 AGACACCCTG GAATGCAAAG TCAAACGGCC TTAGAAAGCC TCACTATGAA CAAAGCTAGTTCTGTGGGAC CTTACGTTTC AGTTTGCCGG AATCTTTCGG AGTGATACTT GTTTCGATCA661 GGAGGTAATG GAATTCCAGT TGAGCTATTT CAAATCTTAA AAGGTGATGC TGTGAAAGTGCCTCCATTAC CTTAAGGTCA ACTCGATAAA GTTTAGAATT TTCCACTACG ACACTTTCAC721 CTGCACTCAA TATGTCAGCA AATTTGGAAA ACTCAGCAGT GGCCACAGGA CTGCCACAATGACGTGAGTT ATACAGTCGT TTAAACCTTT TGAGTCGTCA CCGGTGTCCT GACGGTGTTA781 CCCAAAGAAA AGCAATGACA AAGAATGTTC AAACACCCAC ATGATTGCAC TCATCTCACAGGGTTTCTTT TCGTTACTGT TTCTTACAAG TTTGTGGGTG TACTAACGTG AGTAGAGTGT841 TGCTAGCAAA ATAACTCTCA AAATTCTCCA AGCCAGGCTC CAACAGTACG TGGACCATGAACGATCGTTT TATTGAGAGT TTTAAGAGGT TCGGTCCGAG GTTGTCATGC ACCTGGTACT901 ACTTCCAGAT GTTCAAGCTG GATTTAGAAA AGGCAGAGGA ACCAGAGATC AAATTGCCAATGAAGGTCTA CAAGTTCGAC CTAAATCTTT TCCGTCTCCT TGGTCTCTAG TTTAACGGTT961 CATCCATTGG ATCATCAAAA AAGCACGAGA GTTCCAGAAA AACATCTGCT TTATTGACTAGTAGGTAACC TAGTAGTTTT TTCGTGCTCT CAAGGTCTTT TTGTAGACGA AATAACTGAT1021 CGCTAAAGCC TTTGATTGTG TGGATCACAA TAAACTGTGG AAAATTCTTC AAGAGATGGGGCGATTTCGG AAACTAACAC ACCTAGTGTT ATTTGACACC TTTTAAGAAG TTCTCTACCC1081 AATACCAGAC CACTTTACCT GCCTCCTGAG AAATCTGTAT ACAGGTCCAG AAGCAGCAGTTTATGGTCTG GTGAAATGGA CGGAGGACTC TTTAGACATA TGTCCAGGTC TTCGTCGTCA1141 TAGAACTGGA CATGGAACAA CAGACTGGTT CCAAACTGCG AAAGGGGTAC ATCAAGGAATATCTTGACCT GTACCTTGTT GTCTGACCAA GGTTTGACGC TTTCCCCATG TAGTTCCTTA1201 ATTCATTGGA AGGATTGATG CTGAAGCTGA AACTCCTATA CTTTGGCCAC CTAATGTGAATAAGTAACCT TCCTAACTAC GACTTCGACT TTGAGGATAT GAAACCGGTG GATTACACTT1261 GATCTGACTC ATTGGAAAAG ACTCCAATGC TGGGAAAGAT TGAAGGCAGG AGAAGAGGATCTAGACTGAG TAACCTTTTC TGAGGTTACG ACCCTTTCTA ACTTCCGTCC TCTTCTCCTA1321 GACAGAGGAT GAGATGGTTG GATGGGATCA CTGACTCAAT GGACATGAGT TTGAGTAAGCCTGTCTCCTA CTCTACCAAC CTACCCTAGT GACTGAGTTA CCTGTACTCA AACTCATTCG1381 TCCAGGGGTT GGTGGTGGAC AGGAAAGCCT GGCGTGCTGC AGTCCACAAG GTCACAAAGAAGGTCCCCAA CCACCACCTG TCCTTTCGGA CCGCACGACG TCAGGTGTTC CAGTGTTTCT1441 TTCGGACATG ACTGAGTGAC TGAACTGATA CTGATGTGCT CAACAAATGT ATCTTGAACTAAGCCTGTAC TGACTCACTG ACTTGACTAT GACTACACGA GTTGTTTACA TAGAACTTGA1501 TGTGTGAAGT TCTATGGTCA CATGTAAAGG AAGAATAATC AGGATTAGCT GTGTGTCTTAACACACTTCA AGATACCAGT GTACATTTCC TTCTTATTAG TCCTAATCGA CACACAGAAT1561 GGAATCAGGG TTCTGAGTTT TATGTGTTCA TAGTATCTGC TGGTTCACAA AACATTTTTCCCTTAGTCCC AAGACTCAAA ATACACAAGT ATCATAGACG ACCAAGTGTT TTGTAAAAAG1621 TTATTCTCTG GTTCTTGATT TACTTTATAA AGTAATCTTA ATAGTTATAC TTCACATAGAAATAAGAGAC CAAGAACTAA ATGAAATATT TCATTAGAAT TATCAATATG AAGTGTATCT1681 TACGAAATTA TTATATTTGG ATAATCTCAT GGAAAGGATT AAATACTCCA TCTATTACGAATGCTTTAAT AATATAAACC TATTAGAGTA CCTTTCCTAA TTTATGAGGT AGATAATGCT1741 GTAATGCTGA ACTATCTACT CCTACCTAAT AATTTGTCAG AATTCACTAA TTCTGTGTTACATTACGACT TGATAGATGA GGATGGATTA TTAAACAGTC TTAAGTGATT AAGACACAAT1801 TATTGTTTCT AAATCTGAAT CATTATATGA ATCCTCAGTA TTTTGTTTTC CTTCCTCTATATAACAAAGA TTTAGACTTA GTAATATACT TAGGAGTCAT AAAACAAAAG GAAGGAGATA1861 ATTTTGGAAT TTATTAAACA GTGCTTCAAA TAATTTTTAG GAAACTGAAG TTTTTAGTAATAAAACCTTA AATAATTTGT CACGAAGTTT ATTAAAAATC CTTTGACTTC AAAAATCATT1921 CAGCTCTATC TCTAAATAGC TTTAGTATCT TGAAAAAGTA ATACAAATTC TCACATCCTTGTCGAGATAG AGATTTATCG AAATCATAGA ACTTTTTCAT TATGTTTAAG AGTGTAGGAA1981 AATTTCCTCT TCTCTAAAAT ATCTTTAAAA TATTCTATGA ATGATATCTC TTAATATTTATTAAAGGAGA AGAGATTTTA TAGAAATTTT ATAAGATACT TACTATAGAG AATTATAAAT2041 TTTTTTTGGC AATCCAACAC AGCTTATGGG ATCTTAGTTC CCCAGTGAGG GATTATATCCAAAAAAACCG TTAGGTTGTG TCGAATACCC TAGAATCAAG GGGTCACTCC CTAATATAGG2101 ATGCCAACTG CAGTGAAAGT ACAAAATCCT AAACTGGACT CACCAGGGAT TTCCCAATATTACGGTTGAC GTCACTTTCA TGTTTTAGGA TTTGACCTGA GTGGTCCCTA AAGGGTTATA2161 CTCCTCTAGT TCTTATTTCT GAATATTTTT GGTCCCTTTA TTGTACTCTT CATCCAACTTGAGGAGATCA AGAATAAAGA CTTATAAAAA CCAGGGAAAT AACATGAGAA GTAGGTTGAA2221 TTCTATTGAT TTCTTTCTTG AGGTTATTAT TTACTTGGTT TCAGTTAGAA ATATATGCAAAAGATAACTA AAGAAAGAAC TCCAATAATA AATGAACCAA AGTCAATCTT TATATACGTT2281 ATCTCAGGAC TGCATATTTC AGATTCATTG GCCAATATGG GAAAAAACCT TTGGCTGAACTAGAGTCCTG ACGTATAAAG TCTAAGTAAC CGGTTATACC CTTTTTTGGA AACCGACTTG2341 AAATCATGCT TATAAAAAAT AGTACTAGAG CATCCTACTT TGACTATATC TTGCTCCTCATTTAGTACGA ATATTTTTTA TCATGATCTC GTAGGATGAA ACTGATATAG AACGAGGAGT2401 TTCAGGGTTA TCTAATACAA TTTCCCCACA TGAAATTCTT TTGCATTATA AAAATGGAAGAAGTCCCAAT AGATTATGTT AAAGGGGTGT ACTTTAAGAA AACGTAATAT TTTTACCTTC2461 CTCTTAGGTA ACATTGCAAA AATTCGAGTT GCTCATATGG CACTTTGCTT CTTACTGGTCGAGAATCCAT TGTAACGTTT TTAAGCTCAA CGAGTATACC GTGAAACGAA GAATGACCAG2521 ATTGTGTTCT GAGGCTTACC TGGACAGGTG GTACCTGATG TCATCTTAAA TTGCTGGCTTTAACACAAGA CTCCGAATGG ACCTGTCCAC CATGGACTAC AGTAGAATTT AACGACCGAA2581 TTTGATTTTC CATTGGACAA GCTTCTTTCT TTAGTATATT GTTAAGGATT TCCTTGATCAAAACTAAAAG GTAACCTGTT CGAAGAAAGA AATCATATAA CAATTCCTAA AGGAACTAGT2641 AGATTTTACC TACTTTTCTG GTCCAATTGG TGAGAGACAG TCATAAGGAA ATGCTGTGTTTCTAAAATGG ATGAAAAGAC CAGGTTAACC ACTCTCTGTC AGTATTCCTT TACGACACAA2701 TATTGCACAA TATGTAAAGC ATCTTCCTGA GAAAATAAAA GGGAAATGTT GAATGGGAAGATAACGTGTT ATACATTTCG TAGAAGGACT CTTTTATTTT CCCTTTACAA CTTACCCTTC2761 GATATGCTTT CTTTTGTATT CCTTTTCTGA GAAATCAAAC TTTTTCACCT GTGGCCTTGGCTATACGAAA GAAAACATAA GGAAAAGACT CTTTAGTTTG AAAAAGTGGA CACCGGAACC2821 CCACCAAAAG CTAACAAATA AAGGCATATG AAGTAGCCAA GGCCTTTTCT AGTTATATCTGGTGGTTTTC GATTGTTTAT TTCCGTATAC TTCATCGGTT CCGGAAAAGA TCAATATAGA2881 ATAACACTGA GTTCATTTCA TCATTTATTT TCCTGACTTC CTCCTGGGTC CATATGAGCATATTGTGACT CAAGTAAAGT AGTAAATAAA AGGACTGAAG GAGGACCCAG GTATACTCGT2941 GTCTTAGAAT GAATATTAGC TGAATAATCC AAATACATAG TAGATGTTGA TTTGGGTTTTCAGAATCTTA CTTATAATCG ACTTATTAGG TTTATGTATC ATCTACAACT AAACCCAAAA3001 CTAAGCAATC CAAGACTTGT ATGACAGTAA GATGTATTAC CATCCAACAC ACATCTCAGCGATTCGTTAG GTTCTGAACA TACTGTCATT CTACATAATG GTAGGTTGTG TGTAGAGTCG3061 ATGATATAAA TGCAAGGTAT ATTGTGAAGA AAAATTTTTA ATTATGTCAA AGTGCTTACTTACTATATTT ACGTTCCATA TAACACTTCT TTTTAAAAAT TAATACAGTT TCACGAATGA3121 TTAGAAGGTC ATCTATCTGT CCCAAAGCTG TGAATATATA TATTGAAGGT AATGAATAGAAATCTTCCAG TAGATAGACA GGGTTTCGAC ACTTATATAT ATAACTTCCA TTACTTATCT3181 TGAAGCTAAC CTTGTAAAAA TGAGTAGTGT GAAATACAAC TACAATTATG AACATCTGTCACTTCGATTG GAACATTTTT ACTCATCACA CTTTATGTTG ATGTTAATAC TTGTAGACAG3241 ACTAAAGAGG CAAAGAAACT TGAAGATTGC TTTTGCAAAT GGGCTCCTAT TAATAAAAAGTGATTTCTCC GTTTCTTTGA ACTTCTAACG AAAACGTTTA CCCGAGGATA ATTATTTTTC3301 TACTTTTGAG GTCTGGCTCA GACTCTATTG TAGTACTTAG GGTAAGACCC TCCTCCTGTAATGAAAACTC CAGACCGAGT CTGAGATAAC ATCATGAATC CCATTCTGGG AGGAGGACAT3361 TGGGCTTTCA TTTTCTTTCT TGCTTCCCTC ATTTGCCCTT CCATGAATAC TAGCTGATAAACCCGAAAGT AAAAGAAAGA ACGAAGGGAG TAAACGGGAA GGTACTTATG ATCGACTATT3421 ACATTGACTA TAAAAGATAT GAGGCCAAAC TTGAGCTGTC CCATTTTAAT AAATCTGTATTGTAACTGAT ATTTTCTATA CTCCGGTTTG AACTCGACAG GGTAAAATTA TTTAGACATA3481 AAATAATATT TGTTCTACAA AAGTATTATC TAAATAAATG TTACTTTCTG TCTTAAAATCTTTATTATAA ACAAGATGTT TTCATAATAG ATTTATTTAC AATGAAAGAC AGAATTTTAG3541 CCTCAACAAA TCCCCACTAT CTAGAGAATA AGATTGACAT TCCCTGGAAT CACAGCATGCGGAGTTGTTT AGGGGTGATA GATCTCTTAT TCTAACTGTA AGGGACCTTA GTGTCGTACG3601 TTTGTCTGCC ATTATCTGAC CCCTTTCTCT TTCTCTCTTC TCACCTCCAT CTACTCCTTTAAACAGACGG TAATAGACTG GGGAAAGAGA AAGAGAGAAG AGTGGAGGTA GATGAGGAAA3661 TTCCTTGCAA TTCATGACCC AGATTCACTG TTTGATTTGG CTTGCATGTG TGTGTGCTGAAAGGAACGTT AAGTACTGGG TCTAAGTGAC AAACTAAACC GAACGTACAC ACACACGACT3721 GTTGCGTCTG ACTGTTATCA ACCCCATGAA TGATAGTCCA CCAGGCTCTA CTGTCCATGACAACGCAGAC TGACAATAGT TGGGGTACTT ACTATCAGGT GGTCCGAGAT GACAGGTACT3781 AATTTTCCAG TCAAGAATAC TGGAGTGGAT TGCATTTCCT ACTCCATTTG ATTAATTTAGTTAAAAGGTC AGTTCTTATG ACCTCACCTA ACGTAAAGGA TGAGGTAAAC TAATTAAATC3841 TGACTTTTAA ATTTCTTTTT CCATATTCGG GAGCCTATTC TTCCTTTTTA GTCTATACTCACTGAAAATT TAAAGAAAAA GGTATAAGCC CTCGGATAAG AAGGAAAAAT CAGATATGAG3901 TCTTCACTCT TCAGGTCTAA GGTATCATCG TGTGCTTGTT AGCTTGTTAC TTTCTCCATTAGAAGTGAGA AGTCCAGATT CCATAGTAGC ACACGAACAA TCGAACAATG AAAGAGGTAA3961 ATAGCTTAAG CACTAACAAC TGTTCAGGTT GGCATGAAAT TGTGTTCTTT GTGTGGCCTGTATCGAATTC GTGATTGTTG ACAAGTCCAA CCGTACTTTA ACACAAGAAA CACACCGGAC4021 TATATTTCTG TTGTGTATTA GAATTTACCC CAAGATCTCA AAGACCCACT GAATACTAAAATATAAAGAC AACACATAAT CTTAAATGGG GTTCTAGAGT TTCTGGGTGA CTTATGATTT4081 GAGACCTCAT TGTGGTTACA ATAATTTGGG GACTGGGCCA AAACTACCGT GCATCCCAGCCTCTGGAGTA ACACCAATGT TATTAAACCC CTGACCCGGT TTTGATGGCA CGTAGGGTCG4141 CAAGATCTGT AGCTACTGGA CAATTTCATT TCCTTTATCA GATTGTGAGT TATTCCTGTTGTTCTAGACA TCGATGACCT GTTAAAGTAA AGGAAATAGT CTAACACTCA ATAAGGACAA4201 AAAATGCTCC CCAGAATTTC TGGGGACAGA AAAATAGGAA GAATTCATTT CCTAATCATGTTTTACGAGG GGTCTTAAAG ACCCCTGTCT TTTTATCCTT CTTAAGTAAA GGATTAGTAC4261 CAGATTTCTA GGAATTCAAA TCCACTGTTG GTTTTATTTC AAACCACAAA ATTAGCATGCGTCTAAAGAT CCTTAAGTTT AGGTGACAAC CAAAATAAAG TTTGGTGTTT TAATCGTACG4321 CATTAAATAC TATATATAAA CAGCCACTAA ATCAGATCAT TATCCATTCA GCTTCTCCTTGTAATTTATG ATATATATTT GTCGGTGATT TAGTCTAGTA ATAGGTAAGT CGAAGAGGAA4381 CACTTCTTCT CCTCTACTTT GGAAAAAAGG TAAGAATCTC AGATATAATT TCAGGTGTATGTGAAGAAGA GGAGATGAAA CCTTTTTTCC ATTCTTAGAG TCTATATTAA AGTCCACATA4441 CTGCTACTCA TCTTTATTTT GGACTAGGTT AAAATGTAGA AAGAACATAA TTGCTTAAAAGACGATGAGT AGAAATAAAA CCTGATCCAA TTTTACATCT TTCTTGTATT AACGAATTTT4501 TAGATCTTAA AAATAAGGGT GTTTAAGATA AGGTTTACAC TATTTTCAGC AGATATGTTAATCTAGAATT TTTATTCCCA CAAATTCTAT TCCAAATGTG ATAAAAGTCG TCTATACAAT4561 AAAAATAGAA GTGACTATAA AGACTTGATA AAAATTATAG TGACTGCAAA TGTTTTAGGATTTTTATCTT CACTGATATT TCTGAACTAT TTTTAATATC ACTGACGTTT ACAAAATCCT4621 ATATAATAAG ATATAATAAC GGTGGTTGCT ATTTTCTTTA GCACAAGACT AGTTAACAGGTATATTATTC TATATTATTG CCACCAACGA TAAAAGAAAT CGTGTTCTGA TCAATTGTCC4681 CTGTATTAAA AGATCTTTTC TTGAATTAAA TATTTTCAAT TTGATTAAAC CTACCTCAGCGACATAATTT TCTAGAAAAG AACTTAATTT ATAAAAGTTA AACTAATTTG GATGGAGTCG4741 CATAAAGGCA AGCACATTTC ATTTATACTA TGGGGATTTG AATAATTATT ACTGAAGAAGGTATTTCCGT TCGTGTAAAG TAAATATGAT ACCCCTAAAC TTATTAATAA TGACTTCTTC4801 CTCTACCAAC AAAAAGTTTA TAGAGCTATC ATATTTAGTC AAGAGATAAA GAGGGTTGTTGAGATGGTTG TTTTTCAAAT ATCTCGATAG TATAAATCAG TTCTCTATTT CTCCCAACAA4861 AGGATATATA TGCTATTTGA AAGGTATTTA TAAAAGAAGA GTATATTTAT CAAAATTTCTTCCTATATAT ACGATAAACT TTCCATAAAT ATTTTCTTCT CATATAAATA GTTTTAAAGA4921 CAGAACATCC AAATTTCAAG TTTATCATTT ATCTTACAAT ATTTCAAAAA TATTAAAATAGTCTTGTAGG TTTAAAGTTC AAATAGTAAA TAGAATGTTA TAAAGTTTTT ATAATTTTAT4981 GATACTGAAA TACAGAAGTA AATTAAAGAG AAAGTATTTT ACTTGGTAAA AAAATTCTAGCTATGACTTT ATGTCTTCAT TTAATTTCTC TTTCATAAAA TGAACCATTT TTTTAAGATC5041 GTTGGACAGA GAGTGCCAGG AAACAAAAAC AATGAAAAAT GTGACCTGAC AGGAATTATACAACCTGTCT CTCACGGTCC TTTGTTTTTG TTACTTTTTA CACTGGACTG TCCTTAATAT5101 GCTCAAAGTA TAGTAGTAAG TAATGAAATG GCTTAAAAAT TGGTATATAA AATGCTAGTTCGAGTTTCAT ATCATCATTC ATTACTTTAC CGAATTTTTA ACCATATATT TTACGATCAA5161 ATAAAATAAA CAAAATGCAA TAATATCCTC CCTACATGTA ATGAATTCTA GGTATTATGCTATTTTATTT GTTTTACGTT ATTATAGGAG GGATGTACAT TACTTAAGAT CCATAATACG5221 TCTTTTTGGA AGTCTTGACA ATAAAAATTT TTTTAGAAGT TTATAGGCAT CTTGAATAAAAGAAAAACCT TCAGAACTGT TATTTTTAAA AAAATCTTCA AATATCCGTA GAACTTATTT5281 GTGAAACAAA TTAAGAATTA GTATCCATGA GAAAAATATA GAACAATTTT CCTAATTTAGCACTTTGTTT AATTCTTAAT CATAGGTACT CTTTTTATAT CTTGTTAAAA GGATTAAATC5341 TTTGAAAATC TGGGATTGAA GATGTGTGTC AAGAGATGTT GGTGGCAAGA ACATTTTTTTAAACTTTTAG ACCCTAACTT CTACACACAG TTCTCTACAA CCACCGTTCT TGTAAAAAAA5401 TTCAAGAACT TATAAAAATG CAACAAAACA AACCATTTAA TACATTTTGG TCAAAATCAAAAGTTCTTGA ATATTTTTAC GTTGTTTTGT TTGGTAAATT ATGTAAAACC AGTTTTAGTT5461 TAATGTATTT TATTTTATGC TCCAAGGAGC ATAAAATTGG GGACTGGGCA AGAGAAACTGATTACATAAA ATAAAATACG AGGTTCCTCG TATTTTAACC CCTGACCCGT TCTCTTTGAC5521 ACACCCTGGT AAATTACCAA GAGATAAGTA CACAGTTCTA TGTAGAGAAA ATAAGCATAGTGTGGGACCA TTTAATGGTT CTCTATTCAT GTGTCAAGAT ACATCTCTTT TATTCGTATC5581 TGTATGATCT CTAAAATTAT GTGAGACAAA GGAGAGATGA CATTAGGCAT GTGGGGATGAACATACTAGA GATTTTAATA CACTCTGTTT CCTCTCTACT GTAATCCGTA CACCCCTACT5641 AGACTGAGTA GAGAAGAAAC AATCTAATCA GTCCAAGAAA ACATCTCGAT CAGTGGAACATCTGACTCAT CTCTTCTTTG TTAGATTAGT CAGGTTCTTT TGTAGAGCTA GTCACCTTGT5701 AATAGAAGAA ATGCTAAAAT GAAACAGAAG TCTTACTGGA AATAAAAGAT ATGCATAAGATTATCTTCTT TACGATTTTA CTTTGTCTTC AGAATGACCT TTATTTTCTA TACGTATTCT5761 CAAAAATTCA TGAAAATCAC TTAGTTTAGC AGAGAAAAGA TAAAAATAAA GTATGACCTTGTTTTTAAGT ACTTTTAGTG AATCAAATCG TCTCTTTTCT ATTTTTATTT CATACTGGAA5821 CTTCATATAC ATTGTTTGAT CATATGCACC TCAATAAAAC TGAGTCTCCA ACAGAAATGAGAAGTATATG TAACAAACTA GTATACGTGG AGTTATTTTG ACTCAGAGGT TGTCTTTACT5881 AACATTAATA TTTTGTTCAC TGCTCTAATC CCAGAATCTA AGCGATATCT GGCAATAAAATTGTAATTAT AAAACAAGTG ACGAGATTAG GGTCTTAGAT TCGCTATAGA CCGTTATTTT5941 ATAATAAATA TATATTTTTT AATAAATGAA TCAACCACTT AATTTTTCTG TAAATATCTGTATTATTTAT ATATAAAAAA TTATTTACTT AGTTGGTGAA TTAAAAAGAC ATTTATAGAC6001 TAACTTCTCT TCTGTCTTTC CAAAAACACT CATAAGTACT GTGAATGAGA TGAAAAAGAGATTGAAGAGA AGACAGAAAG GTTTTTGTGA GTATTCATGA CACTTACTCT ACTTTTTCTC6061 TGAAGTAGGA TATAGGCTGT TAGCAGAAAA CATCTGAATG GCTGGCAGTG AAACATTAACACTTCATCCT ATATCCGACA ATCGTCTTTT GTAGACTTAC CGACCGTCAC TTTGTAATTG6121 TTGAAATGTA AGATTAATGA GTAATAGTAA ATTTTAACCT TGGCCATATG ATAAAATGTTAACTTTACAT TCTAATTACT CATTATCATT TAAAATTGGA ACCGGTATAC TATTTTACAA6181 CATTAATATT TTTCTAGAAT ACAGGGCTTT TTGTTTTTGC CATGAGGTTT GCAGGATCTTGTAATTATAA AAAGATCTTA TGTCCCGAAA AACAAAAACG GTACTCCAAA CGTCCTAGAA6241 GGTTCCCTGA CCAGGGATCA AACCTGCACT CCCCTGGAAG CATGGAGTCT TGGACATTTGCCAAGGGACT GGTCCCTAGT TTGGACGTGA GGGGACCTTC GTACCTCAGA ACCTGTAAAC6301 TATTATACAC TATCTTTGGT TCCTTTTAAA GGGAAGTAAT TTTACTTAAA TAAGAAAATAATAATATGTG ATAGAAACCA AGGAAAATTT CCCTTCATTA AAATGAATTT ATTCTTTTAT6361 GATTGACAAG TAATACGCTG TTTCCTCATC TTCCCATTCA CAGGAATCGA GAGCCATGAACTAACTGTTC ATTATGCGAC AAAGGAGTAG AAGGGTAAGT GTCCTTAGCT CTCGGTACTT6421 GGTCCTCATC CTTGCCTGTC TGGTGGCTCT GGCCATTGCG ATCGCCCAGGAGTAG GAACGGACAG ACCACCGAGA CCGGTAACGC TAGCG
3.根據(jù)權(quán)利要求1所述的關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體，其特征在于將關(guān)中奶山羊β-酪蛋白基因啟動區(qū)域序列全長6465bp插入Clontech公司的商售質(zhì)粒pcDNA3.1(-)得到的關(guān)中奶山羊酪蛋白基因啟動子表達(dá)載體全長為11838bp的序列如下1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATGCTGCCTAGCC CTCTAGAGGG CTAGGGGATA CCACGTGAGA GTCATGTTAG ACGAGACTAC61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCGGGCGTATCAA TTCGGTCATA GACGAGGGAC GAACACACAA CCTCCAGCGA CTCATCACGC121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGCGCTCGTTTTA AATTCGATGT TGTTCCGTTC CGAACTGGCT GTTAACGTAC TTCTTAGACG181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATTAATCCCAATC CGCAAAACGC GACGAAGCGC TACATGCCCG GTCTATATGC GCAACTGTAA241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATACTAATAACTG ATCAATAATT ATCATTAGTT AATGCCCCAG TAATCAAGTA TCGGGTATAT301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACCACCTCAAGGC GCAATGTATT GAATGCCATT TACCGGGCGG ACCGACTGGC GGGTTGCTGG361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCCGGGCGGGTAA CTGCAGTTAT TACTGCATAC AAGGGTATCA TTGCGGTTAT CCCTGAAAGG421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGTTAACTGCAGT TACCCACCTC ATAAATGCCA TTTGACGGGT GAACCGTCAT GTAGTTCACA481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATTTAGTATACGG TTCATGCGGG GGATAACTGC AGTTACTGCC ATTTACCGGG CGGACCGTAA541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCATACGGGTCAT GTACTGGAAT ACCCTGAAAG GATGAACCGT CATGTAGATG CATAATCAGT601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTGAGCGATAATG GTACCACTAC GCCAAAACCG TCATGTAGTT ACCCGCACCT ATCGCCAAAC661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACCTGAGTGCCCC TAAAGGTTCA GAGGTGGGGT AACTGCAGTT ACCCTCAAAC AAAACCGTGG721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCGTTTTAGTTGC CCTGAAAGGT TTTACAGCAT TGTTGAGGCG GGGTAACTGC GTTTACCCGC781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCACATCCGCACA TGCCACCCTC CAGATATATT CGTCTCGAGA GACCGATTGA TCTCTTGGGT841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGCGACGAATGAC CGAATAGCTT TAATTATGCT GAGTGATATC CCTCTGGGTT CGACCGATCG901 GTTTAAACGG GCCCTCTAGA CAGATGATTT TGCAACCCCC TGCCTCAGGA GACACTGGGACAAATTTGCC CGGGAGATCT GTCTACTAAA ACGTTGGGGG ACGGAGTCCT CTGTGACCCT961 AATTTCCTGA GACATTTTTG ATTCCAAAAG CTGTGCAGTT GGTGCTTCTA CCATCTTCGTTTAAAGGACT CTGTAAAAAC TAAGGTTTTC GACACGTCAA CCACGAAGAT GGTAGAAGCA1021 GGTAGAGGTC AAGGATGCTG CTAAACATTC TACAACACAT TAAGAAAACC CCCACAACAACCATCTCCAG TTCCTACGAC GATTTGTAAG ATGTTGTGTA ATTCTTTTGG GGGTGTTGTT1081 AGAATTCTTC CGCCAAAAAT ATCAATAATA TGAAGGTTGA AAAATACTGG TCTAGCATGTTCTTAAGAAG GCGGTTTTTA TAGTTATTAT ACTTCCAACT TTTTATGACC AGATCGTACA1141 AGTATGTGCT CAATAGCAAG GAGAGAAAAG AAAGCCTTCC TCACTGATTA ATGCAAAGAATCATACACGA GTTATCGTTC CTCTCTTTTC TTTCGGAAGG AGTGACTAAT TACGTTTCTT1201 ATAGAGGAAA ACAATAGAAT GGGAAAGACT AGAGAGCTCT TCAAGCAAAT TAGAGATATCTATCTCCTTT TGTTATCTTA CCCTTTCTGA TCTCTCGAGA AGTTCGTTTA ATCTCTATAG1261 AAGGGAACAT TTCACGCAAA GATGGGCACA ATAAAGGACA GAAATTTTAT GGAGGAGTTGTTCCCTTGTA AAGTGCGTTT CTACCCGTGT TATTTCCTGT CTTTAAAATA CCTCCTCAAC1321 CTGATGGAGA GGGAGGCCTG GCGTGCTGCG ATTCCTGGGG TCGCAAAGAG TCGGACACAAGACTACCTCT CCCTCCGGAC CGCACGACGC TAAGGACCCC AGCGTTTCTC AGCCTGTGTT1381 CTGAGCGACT GAATTGAACT GAACTGAACT GGACAAAGCA GAAGATATTA AGAAGAGGTGGACTCGCTGA CTTAACTTGA CTTGACTTGA CCTGTTTCGT CTTCTATAAT TCTTCTCCAC1441 GTAAGAATAC ACAGAAGAAC AATATAAAAA AGATCTTCAT GACCCAGATA ACCACGATGACATTCTTATG TGTCTTCTTG TTATATTTTT TCTAGAAGTA CTGGGTCTAT TGGTGCTACT1501 TGTGATCACT CACCTAGAGC CAGACACCCT GGAATGCAAA GTCAAACGGC CTTAGAAAGCACACTAGTGA GTGGATCTCG GTCTGTGGGA CCTTACGTTT CAGTTTGCCG GAATCTTTCG1561 CTCACTATGA ACAAAGCTAG TGGAGGTAAT GGAATTCCAG TTGAGCTATT TCAAATCTTAGAGTGATACT TGTTTCGATC ACCTCCATTA CCTTAAGGTC AACTCGATAA AGTTTAGAAT1621 AAAGGTGATG CTGTGAAAGT GCTGCACTCA ATATGTCAGC AAATTTGGAA AACTCAGCAGTTTCCACTAC GACACTTTCA CGACGTGAGT TATACAGTCG TTTAAACCTT TTGAGTCGTC1681 TGGCCACAGG ACTGCCACAA TCCCAAAGAA AAGCAATGAC AAAGAATGTT CAAACACCCAACCGGTGTCC TGACGGTGTT AGGGTTTCTT TTCGTTACTG TTTCTTACAA GTTTGTGGGT1741 CATGATTGCA CTCATCTCAC ATGCTAGCAA AATAACTCTC AAAATTCTCC AAGCCAGGCTGTACTAACGT GAGTAGAGTG TACGATCGTT TTATTGAGAG TTTTAAGAGG TTCGGTCCGA1801 CCAACAGTAC GTGGACCATG AACTTCCAGA TGTTCAAGCT GGATTTAGAA AAGGCAGAGGGGTTGTCATG CACCTGGTAC TTGAAGGTCT ACAAGTTCGA CCTAAATCTT TTCCGTCTCC1861 AACCAGAGAT CAAATTGCCA ACATCCATTG GATCATCAAA AAAGCACGAG AGTTCCAGAATTGGTCTCTA GTTTAACGGT TGTAGGTAAC CTAGTAGTTT TTTCGTGCTC TCAAGGTCTT1921 AAACATCTGC TTTATTGACT ACGCTAAAGC CTTTGATTGT GTGGATCACA ATAAACTGTGTTTGTAGACG AAATAACTGA TGCGATTTCG GAAACTAACA CACCTAGTGT TATTTGACAC1981 GAAAATTCTT CAAGAGATGG GAATACCAGA CCACTTTACC TGCCTCCTGA GAAATCTGTACTTTTAAGAA GTTCTCTACC CTTATGGTCT GGTGAAATGG ACGGAGGACT CTTTAGACAT2041 TACAGGTCCA GAAGCAGCAG TTAGAACTGG ACATGGAACA ACAGACTGGT TCCAAACTGCATGTCCAGGT CTTCGTCGTC AATCTTGACC TGTACCTTGT TGTCTGACCA AGGTTTGACG2101 GAAAGGGGTA CATCAAGGAA TATTCATTGG AAGGATTGAT GCTGAAGCTG AAACTCCTATCTTTCCCCAT GTAGTTCCTT ATAAGTAACC TTCCTAACTA CGACTTCGAC TTTGAGGATA2161 ACTTTGGCCA CCTAATGTGA AGATCTGACT CATTGGAAAA GACTCCAATG CTGGGAAAGATGAAACCGGT GGATTACACT TCTAGACTGA GTAACCTTTT CTGAGGTTAC GACCCTTTCT2221 TTGAAGGCAG GAGAAGAGGA TGACAGAGGA TGAGATGGTT GGATGGGATC ACTGACTCAAAACTTCCGTC CTCTTCTCCT ACTGTCTCCT ACTCTACCAA CCTACCCTAG TGACTGAGTT2281 TGGACATGAG TTTGAGTAAG CTCCAGGGGT TGGTGGTGGA CAGGAAAGCC TGGCGTGCTGACCTGTACTC AAACTCATTC GAGGTCCCCA ACCACCACCT GTCCTTTCGG ACCGCACGAC2341 CAGTCCACAA GGTCACAAAG ATTCGGACAT GACTGAGTGA CTGAACTGAT ACTGATGTGCGTCAGGTGTT CCAGTGTTTC TAAGCCTGTA CTGACTCACT GACTTGACTA TGACTACACG2401 TCAACAAATG TATCTTGAAC TTGTGTGAAG TTCTATGGTC ACATGTAAAG GAAGAATAATAGTTGTTTAC ATAGAACTTG AACACACTTC AAGATACCAG TGTACATTTC CTTCTTATTA2461 CAGGATTAGC TGTGTGTCTT AGGAATCAGG GTTCTGAGTT TTATGTGTTC ATAGTATCTGGTCCTAATCG ACACACAGAA TCCTTAGTCC CAAGACTCAA AATACACAAG TATCATAGAC2521 CTGGTTCACA AAACATTTTT CTTATTCTCT GGTTCTTGAT TTACTTTATA AAGTAATCTTGACCAAGTGT TTTGTAAAAA GAATAAGAGA CCAAGAACTA AATGAAATAT TTCATTAGAA2581 AATAGTTATA CTTCACATAG ATACGAAATT ATTATATTTG GATAATCTCA TGGAAAGGATTTATCAATAT GAAGTGTATC TATGCTTTAA TAATATAAAC CTATTAGAGT ACCTTTCCTA2641 TAAATACTCC ATCTATTACG AGTAATGCTG AACTATCTAC TCCTACCTAA TAATTTGTCAATTTATGAGG TAGATAATGC TCATTACGAC TTGATAGATG AGGATGGATT ATTAAACAGT2701 GAATTCACTA ATTCTGTGTT ATATTGTTTC TAAATCTGAA TCATTATATG AATCCTCAGTCTTAAGTGAT TAAGACACAA TATAACAAAG ATTTAGACTT AGTAATATAC TTAGGAGTCA2761 ATTTTGTTTT CCTTCCTCTA TATTTTGGAA TTTATTAAAC AGTGCTTCAA ATAATTTTTATAAAACAAAA GGAAGGAGAT ATAAAACCTT AAATAATTTG TCACGAAGTT TATTAAAAAT2821 GGAAACTGAA GTTTTTAGTA ACAGCTCTAT CTCTAAATAG CTTTAGTATC TTGAAAAAGTCCTTTGACTT CAAAAATCAT TGTCGAGATA GAGATTTATC GAAATCATAG AACTTTTTCA2881 AATACAAATT CTCACATCCT TAATTTCCTC TTCTCTAAAA TATCTTTAAA ATATTCTATGTTATGTTTAA GAGTGTAGGA ATTAAAGGAG AAGAGATTTT ATAGAAATTT TATAAGATAC2941 AATGATATCT CTTAATATTT ATTTTTTTGG CAATCCAACA CAGCTTATGG GATCTTAGTTTTACTATAGA GAATTATAAA TAAAAAAACC GTTAGGTTGT GTCGAATACC CTAGAATCAA3001 CCCCAGTGAG GGATTATATC CATGCCAACT GCAGTGAAAG TACAAAATCC TAAACTGGACGGGGTCACTC CCTAATATAG GTACGGTTGA CGTCACTTTC ATGTTTTAGG ATTTGACCTG3061 TCACCAGGGA TTTCCCAATA TCTCCTCTAG TTCTTATTTC TGAATATTTT TGGTCCCTTTAGTGGTCCCT AAAGGGTTAT AGAGGAGATC AAGAATAAAG ACTTATAAAA ACCAGGGAAA3121 ATTGTACTCT TCATCCAACT TTTCTATTGA TTTCTTTCTT GAGGTTATTA TTTACTTGGTTAACATGAGA AGTAGGTTGA AAAGATAACT AAAGAAAGAA CTCCAATAAT AAATGAACCA3181 TTCAGTTAGA AATATATGCA AATCTCAGGA CTGCATATTT CAGATTCATT GGCCAATATGAAGTCAATCT TTATATACGT TTAGAGTCCT GACGTATAAA GTCTAAGTAA CCGGTTATAC3241 GGAAAAAACC TTTGGCTGAA CAAATCATGC TTATAAAAAA TAGTACTAGA GCATCCTACTCCTTTTTTGG AAACCGACTT GTTTAGTACG AATATTTTTT ATCATGATCT CGTAGGATGA3301 TTGACTATAT CTTGCTCCTC ATTCAGGGTT ATCTAATACA ATTTCCCCAC ATGAAATTCTAACTGATATA GAACGAGGAG TAAGTCCCAA TAGATTATGT TAAAGGGGTG TACTTTAAGA3361 TTTGCATTAT AAAAATGGAA GCTCTTAGGT AACATTGCAA AAATTCGAGT TGCTCATATGAAACGTAATA TTTTTACCTT CGAGAATCCA TTGTAACGTT TTTAAGCTCA ACGAGTATAC3421 GCACTTTGCT TCTTACTGGT CATTGTGTTC TGAGGCTTAC CTGGACAGGT GGTACCTGATCGTGAAACGA AGAATGACCA GTAACACAAG ACTCCGAATG GACCTGTCCA CCATGGACTA3481 GTCATCTTAA ATTGCTGGCT TTTTGATTTT CCATTGGACA AGCTTCTTTC TTTAGTATATCAGTAGAATT TAACGACCGA AAAACTAAAA GGTAACCTGT TCGAAGAAAG AAATCATATA3541 TGTTAAGGAT TTCCTTGATC AAGATTTTAC CTACTTTTCT GGTCCAATTG GTGAGAGACAACAATTCCTA AAGGAACTAG TTCTAAAATG GATGAAAAGA CCAGGTTAAC CACTCTCTGT3601 GTCATAAGGA AATGCTGTGT TTATTGCACA ATATGTAAAG CATCTTCCTG AGAAAATAAACAGTATTCCT TTACGACACA AATAACGTGT TATACATTTC GTAGAAGGAC TCTTTTATTT3661 AGGGAAATGT TGAATGGGAA GGATATGCTT TCTTTTGTAT TCCTTTTCTG AGAAATCAAATCCCTTTACA ACTTACCCTT CCTATACGAA AGAAAACATA AGGAAAAGAC TCTTTAGTTT3721 CTTTTTCACC TGTGGCCTTG GCCACCAAAA GCTAACAAAT AAAGGCATAT GAAGTAGCCAGAAAAAGTGG ACACCGGAAC CGGTGGTTTT CGATTGTTTA TTTCCGTATA CTTCATCGGT3781 AGGCCTTTTC TAGTTATATC TATAACACTG AGTTCATTTC ATCATTTATT TTCCTGACTTTCCGGAAAAG ATCAATATAG ATATTGTGAC TCAAGTAAAG TAGTAAATAA AAGGACTGAA3841 CCTCCTGGGT CCATATGAGC AGTCTTAGAA TGAATATTAG CTGAATAATC CAAATACATAGGAGGACCCA GGTATACTCG TCAGAATCTT ACTTATAATC GACTTATTAG GTTTATGTAT3901 GTAGATGTTG ATTTGGGTTT TCTAAGCAAT CCAAGACTTG TATGACAGTA AGATGTATTACATCTACAAC TAAACCCAAA AGATTCGTTA GGTTCTGAAC ATACTGTCAT TCTACATAAT3961 CCATCCAACA CACATCTCAG CATGATATAA ATGCAAGGTA TATTGTGAAG AAAAATTTTTGGTAGGTTGT GTGTAGAGTC GTACTATATT TACGTTCCAT ATAACACTTC TTTTTAAAAA4021 AATTATGTCA AAGTGCTTAC TTTAGAAGGT CATCTATCTG TCCCAAAGCT GTGAATATATTTAATACAGT TTCACGAATG AAATCTTCCA GTAGATAGAC AGGGTTTCGA CACTTATATA4081 ATATTGAAGG TAATGAATAG ATGAAGCTAA CCTTGTAAAA ATGAGTAGTG TGAAATACAATATAACTTCC ATTACTTATC TACTTCGATT GGAACATTTT TACTCATCAC ACTTTATGTT4141 CTACAATTAT GAACATCTGT CACTAAAGAG GCAAAGAAAC TTGAAGATTG CTTTTGCAAAGATGTTAATA CTTGTAGACA GTGATTTCTC CGTTTCTTTG AACTTCTAAC GAAAACGTTT4201 TGGGCTCCTA TTAATAAAAA GTACTTTTGA GGTCTGGCTC AGACTCTATT GTAGTACTTAACCCGAGGAT AATTATTTTT CATGAAAACT CCAGACCGAG TCTGAGATAA CATCATGAAT4261 GGGTAAGACC CTCCTCCTGT ATGGGCTTTC ATTTTCTTTC TTGCTTCCCT CATTTGCCCTCCCATTCTGG GAGGAGGACA TACCCGAAAG TAAAAGAAAG AACGAAGGGA GTAAACGGGA4321 TCCATGAATA CTAGCTGATA AACATTGACT ATAAAAGATA TGAGGCCAAA CTTGAGCTGTAGGTACTTAT GATCGACTAT TTGTAACTGA TATTTTCTAT ACTCCGGTTT GAACTCGACA4381 CCCATTTTAA TAAATCTGTA TAAATAATAT TTGTTCTACA AAAGTATTAT CTAAATAAATGGGTAAAATT ATTTAGACAT ATTTATTATA AACAAGATGT TTTCATAATA GATTTATTTA4441 GTTACTTTCT GTCTTAAAAT CCCTCAACAA ATCCCCACTA TCTAGAGAAT AAGATTGACACAATGAAAGA CAGAATTTTA GGGAGTTGTT TAGGGGTGAT AGATCTCTTA TTCTAACTGT4501 TTCCCTGGAA TCACAGCATG CTTTGTCTGC CATTATCTGA CCCCTTTCTC TTTCTCTCTTAAGGGACCTT AGTGTCGTAC GAAACAGACG GTAATAGACT GGGGAAAGAG AAAGAGAGAA4561 CTCACCTCCA TCTACTCCTT TTTCCTTGCA ATTCATGACC CAGATTCACT GTTTGATTTGGAGTGGAGGT AGATGAGGAA AAAGGAACGT TAAGTACTGG GTCTAAGTGA CAAACTAAAC4621 GCTTGCATGT GTGTGTGCTG AGTTGCGTCT GACTGTTATC AACCCCATGA ATGATAGTCCCGAACGTACA CACACACGAC TCAACGCAGA CTGACAATAG TTGGGGTACT TACTATCAGG4681 ACCAGGCTCT ACTGTCCATG AAATTTTCCA GTCAAGAATA CTGGAGTGGA TTGCATTTCCTGGTCCGAGA TGACAGGTAC TTTAAAAGGT CAGTTCTTAT GACCTCACCT AACGTAAAGG4741 TACTCCATTT GATTAATTTA GTGACTTTTA AATTTCTTTT TCCATATTCG GGAGCCTATTATGAGGTAAA CTAATTAAAT CACTGAAAAT TTAAAGAAAA AGGTATAAGC CCTCGGATAA4801 CTTCCTTTTT AGTCTATACT CTCTTCACTC TTCAGGTCTA AGGTATCATC GTGTGCTTGTGAAGGAAAAA TCAGATATGA GAGAAGTGAG AAGTCCAGAT TCCATAGTAG CACACGAACA4861 TAGCTTGTTA CTTTCTCCAT TATAGCTTAA GCACTAACAA CTGTTCAGGT TGGCATGAAAATCGAACAAT GAAAGAGGTA ATATCGAATT CGTGATTGTT GACAAGTCCA ACCGTACTTT4921 TTGTGTTCTT TGTGTGGCCT GTATATTTCT GTTGTGTATT AGAATTTACC CCAAGATCTCAACACAAGAA ACACACCGGA CATATAAAGA CAACACATAA TCTTAAATGG GGTTCTAGAG4981 AAAGACCCAC TGAATACTAA AGAGACCTCA TTGTGGTTAC AATAATTTGG GGACTGGGCCTTTCTGGGTG ACTTATGATT TCTCTGGAGT AACACCAATG TTATTAAACC CCTGACCCGG5041 AAAACTACCG TGCATCCCAG CCAAGATCTG TAGCTACTGG ACAATTTCAT TTCCTTTATCTTTTGATGGC ACGTAGGGTC GGTTCTAGAC ATCGATGACC TGTTAAAGTA AAGGAAATAG5101 AGATTGTGAG TTATTCCTGT TAAAATGCTC CCCAGAATTT CTGGGGACAG AAAAATAGGATCTAACACTC AATAAGGACA ATTTTACGAG GGGTCTTAAA GACCCCTGTC TTTTTATCCT5161 AGAATTCATT TCCTAATCAT GCAGATTTCT AGGAATTCAA ATCCACTGTT GGTTTTATTTTCTTAAGTAA AGGATTAGTA CGTCTAAAGA TCCTTAAGTT TAGGTGACAA CCAAAATAAA5221 CAAACCACAA AATTAGCATG CCATTAAATA CTATATATAA ACAGCCACTA AATCAGATCAGTTTGGTGTT TTAATCGTAC GGTAATTTAT GATATATATT TGTCGGTGAT TTAGTCTAGT5281 TTATCCATTC AGCTTCTCCT TCACTTCTTC TCCTCTACTT TGGAAAAAAG GTAAGAATCTAATAGGTAAG TCGAAGAGGA AGTGAAGAAG AGGAGATGAA ACCTTTTTTC CATTCTTAGA5341 CAGATATAAT TTCAGGTGTA TCTGCTACTC ATCTTTATTT TGGACTAGGT TAAAATGTAGGTCTATATTA AAGTCCACAT AGACGATGAG TAGAAATAAA ACCTGATCCA ATTTTACATC5401 AAAGAACATA ATTGCTTAAA ATAGATCTTA AAAATAAGGG TGTTTAAGAT AAGGTTTACATTTCTTGTAT TAACGAATTT TATCTAGAAT TTTTATTCCC ACAAATTCTA TTCCAAATGT5461 CTATTTTCAG CAGATATGTT AAAAAATAGA AGTGACTATA AAGACTTGAT AAAAATTATAGATAAAAGTC GTCTATACAA TTTTTTATCT TCACTGATAT TTCTGAACTA TTTTTAATAT5521 GTGACTGCAA ATGTTTTAGG AATATAATAA GATATAATAA CGGTGGTTGC TATTTTCTTTCACTGACGTT TACAAAATCC TTATATTATT CTATATTATT GCCACCAACG ATAAAAGAAA5581 AGCACAAGAC TAGTTAACAG GCTGTATTAA AAGATCTTTT CTTGAATTAA ATATTTTCAATCGTGTTCTG ATCAATTGTC CGACATAATT TTCTAGAAAA GAACTTAATT TATAAAAGTT5641 TTTGATTAAA CCTACCTCAG CCATAAAGGC AAGCACATTT CATTTATACT ATGGGGATTTAAACTAATTT GGATGGAGTC GGTATTTCCG TTCGTGTAAA GTAAATATGA TACCCCTAAA5701 GAATAATTAT TACTGAAGAA GCTCTACCAA CAAAAAGTTT ATAGAGCTAT CATATTTAGTCTTATTAATA ATGACTTCTT CGAGATGGTT GTTTTTCAAA TATCTCGATA GTATAAATCA5761 CAAGAGATAA AGAGGGTTGT TAGGATATAT ATGCTATTTG AAAGGTATTT ATAAAAGAAGGTTCTCTATT TCTCCCAACA ATCCTATATA TACGATAAAC TTTCCATAAA TATTTTCTTC5821 AGTATATTTA TCAAAATTTC TCAGAACATC CAAATTTCAA GTTTATCATT TATCTTACAATCATATAAAT AGTTTTAAAG AGTCTTGTAG GTTTAAAGTT CAAATAGTAA ATAGAATGTT5881 TATTTCAAAA ATATTAAAAT AGATACTGAA ATACAGAAGT AAATTAAAGA GAAAGTATTTATAAAGTTTT TATAATTTTA TCTATGACTT TATGTCTTCA TTTAATTTCT CTTTCATAAA5941 TACTTGGTAA AAAAATTCTA GGTTGGACAG AGAGTGCCAG GAAACAAAAA CAATGAAAAAATGAACCATT TTTTTAAGAT CCAACCTGTC TCTCACGGTC CTTTGTTTTT GTTACTTTTT6001 TGTGACCTGA CAGGAATTAT AGCTCAAAGT ATAGTAGTAA GTAATGAAAT GGCTTAAAAAACACTGGACT GTCCTTAATA TCGAGTTTCA TATCATCATT CATTACTTTA CCGAATTTTT6061 TTGGTATATA AAATGCTAGT TATAAAATAA ACAAAATGCA ATAATATCCT CCCTACATGTAACCATATAT TTTACGATCA ATATTTTATT TGTTTTACGT TATTATAGGA GGGATGTACA6121 AATGAATTCT AGGTATTATG CTCTTTTTGG AAGTCTTGAC AATAAAAATT TTTTTAGAAGTTACTTAAGA TCCATAATAC GAGAAAAACC TTCAGAACTG TTATTTTTAA AAAAATCTTC6181 TTTATAGGCA TCTTGAATAA AGTGAAACAA ATTAAGAATT AGTATCCATG AGAAAAATATAAATATCCGT AGAACTTATT TCACTTTGTT TAATTCTTAA TCATAGGTAC TCTTTTTATA6241 AGAACAATTT TCCTAATTTA GTTTGAAAAT CTGGGATTGA AGATGTGTGT CAAGAGATGTTCTTGTTAAA AGGATTAAAT CAAACTTTTA GACCCTAACT TCTACACACA GTTCTCTACA6301 TGGTGGCAAG AACATTTTTT TTTCAAGAAC TTATAAAAAT GCAACAAAAC AAACCATTTAACCACCGTTC TTGTAAAAAA AAAGTTCTTG AATATTTTTA CGTTGTTTTG TTTGGTAAAT6361 ATACATTTTG GTCAAAATCA ATAATGTATT TTATTTTATG CTCCAAGGAG CATAAAATTGTATGTAAAAC CAGTTTTAGT TATTACATAA AATAAAATAC GAGGTTCCTC GTATTTTAAC6421 GGGACTGGGC AAGAGAAACT GACACCCTGG TAAATTACCA AGAGATAAGT ACACAGTTCTCCCTGACCCG TTCTCTTTGA CTGTGGGACC ATTTAATGGT TCTCTATTCA TGTGTCAAGA6481 ATGTAGAGAA AATAAGCATA GTGTATGATC TCTAAAATTA TGTGAGACAA AGGAGAGATGTACATCTCTT TTATTCGTAT CACATACTAG AGATTTTAAT ACACTCTGTT TCCTCTCTAC6541 ACATTAGGCA TGTGGGGATG AAGACTGAGT AGAGAAGAAA CAATCTAATC AGTCCAAGAATGTAATCCGT ACACCCCTAC TTCTGACTCA TCTCTTCTTT GTTAGATTAG TCAGGTTCTT6601 AACATCTCGA TCAGTGGAAC AAATAGAAGA AATGCTAAAA TGAAACAGAA GTCTTACTGGTTGTAGAGCT AGTCACCTTG TTTATCTTCT TTACGATTTT ACTTTGTCTT CAGAATGACC6661 AAATAAAAGA TATGCATAAG ACAAAAATTC ATGAAAATCA CTTAGTTTAG CAGAGAAAAGTTTATTTTCT ATACGTATTC TGTTTTTAAG TACTTTTAGT GAATCAAATC GTCTCTTTTC6721 ATAAAAATAA AGTATGACCT TCTTCATATA CATTGTTTGA TCATATGCAC CTCAATAAAATATTTTTATT TCATACTGGA AGAAGTATAT GTAACAAACT AGTATACGTG GAGTTATTTT6781 CTGAGTCTCC AACAGAAATG AAACATTAAT ATTTTGTTCA CTGCTCTAAT CCCAGAATCTGACTCAGAGG TTGTCTTTAC TTTGTAATTA TAAAACAAGT GACGAGATTA GGGTCTTAGA6841 AAGCGATATC TGGCAATAAA AATAATAAAT ATATATTTTT TAATAAATGA ATCAACCACTTTCGCTATAG ACCGTTATTT TTATTATTTA TATATAAAAA ATTATTTACT TAGTTGGTGA6901 TAATTTTTCT GTAAATATCT GTAACTTCTC TTCTGTCTTT CCAAAAACAC TCATAAGTACATTAAAAAGA CATTTATAGA CATTGAAGAG AAGACAGAAA GGTTTTTGTG AGTATTCATG6961 TGTGAATGAG ATGAAAAAGA GTGAAGTAGG ATATAGGCTG TTAGCAGAAA ACATCTGAATACACTTACTC TACTTTTTCT CACTTCATCC TATATCCGAC AATCGTCTTT TGTAGACTTA7021 GGCTGGCAGT GAAACATTAA CTTGAAATGT AAGATTAATG AGTAATAGTA AATTTTAACCCCGACCGTCA CTTTGTAATT GAACTTTACA TTCTAATTAC TCATTATCAT TTAAAATTGG7081 TTGGCCATAT GATAAAATGT TCATTAATAT TTTTCTAGAA TACAGGGCTT TTTGTTTTTGAACCGGTATA CTATTTTACA AGTAATTATA AAAAGATCTT ATGTCCCGAA AAACAAAAAC7141 CCATGAGGTT TGCAGGATCT TGGTTCCCTG ACCAGGGATC AAACCTGCAC TCCCCTGGAAGGTACTCCAA ACGTCCTAGA ACCAAGGGAC TGGTCCCTAG TTTGGACGTG AGGGGACCTT7201 GCATGGAGTC TTGGACATTT GTATTATACA CTATCTTTGG TTCCTTTTAA AGGGAAGTAACGTACCTCAG AACCTGTAAA CATAATATGT GATAGAAACC AAGGAAAATT TCCCTTCATT7261 TTTTACTTAA ATAAGAAAAT AGATTGACAA GTAATACGCT GTTTCCTCAT CTTCCCATTCAAAATGAATT TATTCTTTTA TCTAACTGTT CATTATGCGA CAAAGGAGTA GAAGGGTAAG7321 ACAGGAATCG AGAGCCATGA AGGTCCTCAT CCTTGCCTGT CTGGTGGCTC TGGCCATTGCTGTCCTTAGC TCTCGGTACT TCCAGGAGTA GGAACGGACA GACCACCGAG ACCGGTAACG7381 GATCGCGGAT CCGAGCTCGG TACCAAGCTT AAGTTTAAAC CCGCTGATCA GCCTCGACTGCTAGCGCCTA GGCTCGAGCC ATGGTTCGAA TTCAAATTTG GGCGACTAGT CGGAGCTGAC7441 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGGACGGAAGATC AACGGTCGGT AGACAACAAA CGGGGAGGGG GCACGGAAGG AACTGGGACC7501 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGATTCCACGGTG AGGGTGACAG GAAAGGATTA TTTTACTCCT TTAACGTAGC GTAACAGACT7561 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGGCATCCACAGT AAGATAAGAC CCCCCACCCC ACCCCGTCCT GTCGTTCCCC CTCCTAACCC7621 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAATTCTGTTATC GTCCGTACGA CCCCTACGCC ACCCGAGATA CCGAAGACTC CGCCTTTCTT7681 CCAGCTGGGG CTCTAGGGGG TATCCCCACG CGCCCTGTAG CGGCGCATTA AGCGCGGCGGGGTCGACCCC GAGATCCCCC ATAGGGGTGC GCGGGACATC GCCGCGTAAT TCGCGCCGCC7741 GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTTCACACCACCA ATGCGCGTCG CACTGGCGAT GTGAACGGTC GCGGGATCGC GGGCGAGGAA7801 TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATCAGCGAAAGAA GGGAAGGAAA GAGCGGTGCA AGCGGCCGAA AGGGGCAGTT CGAGATTTAG7861 GGGGGCTCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTGCCCCCGAGGG AAATCCCAAG GCTAAATCAC GAAATGCCGT GGAGCTGGGG TTTTTTGAAC7921 ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGATAATCCCACT ACCAAGTGCA TCACCCGGTA GCGGGACTAT CTGCCAAAAA GCGGGAAACT7981 CGTTGGAGTC CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA ACACTCAACCGCAACCTCAG GTGCAAGAAA TTATCACCTG AGAACAAGGT TTGACCTTGT TGTGAGTTGG8041 CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC GATTTCGGCC TATTGGTTAAGATAGAGCCA GATAAGAAAA CTAAATATTC CCTAAAACGG CTAAAGCCGG ATAACCAATT8101 AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG TGTGTCAGTTTTTTACTCGA CTAAATTGTT TTTAAATTGC GCTTAATTAA GACACCTTAC ACACAGTCAA8161 AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAATCCCACACCT TTCAGGGGTC CGAGGGGTCG TCCGTCTTCA TACGTTTCGT ACGTAGAGTT8221 TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAGAATCAGTCGT TGGTCCACAC CTTTCAGGGG TCCGAGGGGT CGTCCGTCTT CATACGTTTC8281 CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCTGTACGTAGAG TTAATCAGTC GTTGGTATCA GGGCGGGGAT TGAGGCGGGT AGGGCGGGGA8341 AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA CTAATTTTTT TTATTTATGCTTGAGGCGGG TCAAGGCGGG TAAGAGGCGG GGTACCGACT GATTAAAAAA AATAAATACG8401 AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGGTCTCCGGCTC CGGCGGAGAC GGAGACTCGA TAAGGTCTTC ATCACTCCTC CGAAAAAACC8461 AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA TCCATTTTCG GATCTGATCATCCGGATCCG AAAACGTTTT TCGAGGGCCC TCGAACATAT AGGTAAAAGC CTAGACTAGT8521 AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCCTCTCTGTCCT ACTCCTAGCA AAGCGTACTA ACTTGTTCTA CCTAACGTGC GTCCAAGAGG8581 GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTCCCGGCGAACC CACCTCTCCG ATAAGCCGAT ACTGACCCGT GTTGTCTGTT AGCCGACGAG8641 TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGAACTACGGCGG CACAAGGCCG ACAGTCGCGT CCCCGCGGGC CAAGAAAAAC AGTTCTGGCT8701 CCTGTCCGGT GCCCTGAATG AACTGCAGGA CGAGGCAGCG CGGCTATCGT GGCTGGCCACGGACAGGCCA CGGGACTTAC TTGACGTCCT GCTCCGTCGC GCCGATAGCA CCGACCGGTG8761 GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCTCTGCCCGCAA GGAACGCGTC GACACGAGCT GCAACAGTGA CTTCGCCCTT CCCTGACCGA8821 GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAACGATAACCCG CTTCACGGCC CCGTCCTAGA GGACAGTAGA GTGGAACGAG GACGGCTCTT8881 AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCCTCATAGGTAG TACCGACTAC GTTACGCCGC CGACGTATGC GAACTAGGCC GATGGACGGG8941 ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCTTAAGCTGGTG GTTCGCTTTG TAGCGTAGCT CGCTCGTGCA TGAGCCTACC TTCGGCCAGA9001 TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGCACAGCTAGTC CTACTAGACC TGCTTCTCGT AGTCCCCGAG CGCGGTCGGC TTGACAAGCG9061 CAGGCTCAAG GCGCGCATGC CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTGGTCCGAGTTC CGCGCGTACG GGCTGCCGCT CCTAGAGCAG CACTGGGTAC CGCTACGGAC9121 CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCTGAACGGCTTA TAGTACCACC TTTTACCGGC GAAAAGACCT AAGTAGCTGA CACCGGCCGA9181 GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCTCCCACACCGC CTGGCGATAG TCCTGTATCG CAACCGATGG GCACTATAAC GACTTCTCGA9241 TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCAACCGCCGCTT ACCCGACTGG CGAAGGAGCA CGAAATGCCA TAGCGGCGAG GGCTAAGCGT9301 GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAACGCGTAGCGG AAGATAGCGG AAGAACTGCT CAAGAAGACT CGCCCTGAGA CCCCAAGCTT9361 ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTCTACTGGCTGG TTCGCTGCGG GTTGGACGGT AGTGCTCTAA AGCTAAGGTG GCGGCGGAAG9421 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGCATACTTTCCA ACCCGAAGCC TTAGCAAAAG GCCCTGCGGC CGACCTACTA GGAGGTCGCG9481 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC TTATAATGGTCCCCTAGAGT ACGACCTCAA GAAGCGGGTG GGGTTGAACA AATAACGTCG AATATTACCA9541 TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCTATGTTTATTT CGTTATCGTA GTGTTTAAAG TGTTTATTTC GTAAAAAAAG TGACGTAAGA9601 AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCTTCAACACCAA ACAGGTTTGA GTAGTTACAT AGAATAGTAC AGACATATGG CAGCTGGAGA9661 AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTCTCGATCTCGA ACCGCATTAG TACCAGTATC GACAAAGGAC ACACTTTAAC AATAGGCGAG9721 ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGATGTTAAGGTG TGTTGTATGC TCGGCCTTCG TATTTCACAT TTCGGACCCC ACGGATTACT9781 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTGCACTCGATTG AGTGTAATTA ACGCAACGCG AGTGACGGGC GAAAGGTCAG CCCTTTGGAC9841 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGGAGCACGGTCG ACGTAATTAC TTAGCCGGTT GCGCGCCCCT CTCCGCCAAA CGCATAACCC9901 CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCGGCGAGAAGGC GAAGGAGCGA GTGACTGAGC GACGCGAGCC AGCAAGCCGA CGCCGCTCGC9961 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGACATAGTCGAG TGAGTTTCCG CCATTATGCC AATAGGTGTC TTAGTCCCCT ATTGCGTCCT10021 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTGTTCTTGTACA CTCGTTTTCC GGTCGTTTTC CGGTCCTTGG CATTTTTCCG GCGCAACGAC10081 GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAGCGCAAAAAGG TATCCGAGGC GGGGGGACTG CTCGTAGTGT TTTTAGCTGC GAGTTCAGTC10141 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTCTCCACCGCTT TGGGCTGTCC TGATATTTCT ATGGTCCGCA AAGGGGGACC TTCGAGGGAG10201 GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCGCACGCGAGAG GACAAGGCTG GGACGGCGAA TGGCCTATGG ACAGGCGGAA AGAGGGAAGC10261 GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTTCCTTCGCACC GCGAAAGAGT ATCGAGTGCG ACATCCATAG AGTCAAGCCA CATCCAGCAA10321 CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCCGCGAGGTTCG ACCCGACACA CGTGCTTGGG GGGCAAGTCG GGCTGGCGAC GCGGAATAGG10381 GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCCCCATTGATAG CAGAACTCAG GTTGGGCCAT TCTGTGCTGA ATAGCGGTGA CCGTCGTCGG10441 ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGGTGACCATTGT CCTAATCGTC TCGCTCCATA CATCCGCCAC GATGTCTCAA GAACTTCACC10501 TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCAACCGGATTGA TGCCGATGTG ATCTTCTTGT CATAAACCAT AGACGCGAGA CGACTTCGGT10561 GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGCCAATGGAAGC CTTTTTCTCA ACCATCGAGA ACTAGGCCGT TTGTTTGGTG GCGACCATCG10621 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCTCCAAAAAAAC AAACGTTCGT CGTCTAATGC GCGTCTTTTT TTCCTAGAGT TCTTCTAGGA10681 TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTGAACTAGAAAA GATGCCCCAG ACTGCGAGTC ACCTTGCTTT TGAGTGCAAT TCCCTAAAAC10741 GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTTCAGTACTCTA ATAGTTTTTC CTAGAAGTGG ATCTAGGAAA ATTTAATTTT TACTTCAAAA10801 AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGTTTTAGTTAGA TTTCATATAT ACTCATTTGA ACCAGACTGT CAATGGTTAC GAATTAGTCA10861 GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTCCTCCGTGGAT AGAGTCGCTA GACAGATAAA GCAAGTAGGT ATCAACGGAC TGAGGGGCAG10921 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCGCACATCTATT GATGCTATGC CCTCCCGAAT GGTAGACCGG GGTCACGACG TTACTATGGC10981 CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCCGCTCTGGGTG CGAGTGGCCG AGGTCTAAAT AGTCGTTATT TGGTCGGTCG GCCTTCCCGG11041 GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGGCTCGCGTCTT CACCAGGACG TTGAAATAGG CGGAGGTAGG TCAGATAATT AACAACGGCC11101 GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACACTTCGATCTC ATTCATCAAG CGGTCAATTA TCAAACGCGT TGCAACAACG GTAACGATGT11161 GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGACCGTAGCACC ACAGTGCGAG CAGCAAACCA TACCGAAGTA AGTCGAGGCC AAGGGTTGCT11221 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCTAGTTCCGCTC AATGTACTAG GGGGTACAAC ACGTTTTTTC GCCAATCGAG GAAGCCAGGA11281 CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTGGGCTAGCAAC AGTCTTCATT CAACCGGCGT CACAATAGTG AGTACCAATA CCGTCGTGAC11341 CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCAGTATTAAGAG AATGACAGTA CGGTAGGCAT TCTACGAAAA GACACTGACC ACTCATGAGT11401 ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATATGGTTCAGTA AGACTCTTAT CACATACGCC GCTGGCTCAA CGAGAACGGG CCGCAGTTAT11461 CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCTGCCCTATTAT GGCGCGGTGT ATCGTCTTGA AATTTTCACG AGTAGTAACC TTTTGCAAGA11521 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACTAGCCCCGCTT TTGAGAGTTC CTAGAATGGC GACAACTCTA GGTCAAGCTA CATTGGGTGA11581 CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAAGCACGTGGGT TGACTAGAAG TCGTAGAAAA TGAAAGTGGT CGCAAAGACC CACTCGTTTT11641 ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTCTGTCCTTCCG TTTTACGGCG TTTTTTCCCT TATTCCCGCT GTGCCTTTAC AACTTATGAG11701 ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGATATGAGAAGG AAAAAGTTAT AATAACTTCG TAAATAGTCC CAATAACAGA GTACTCGCCT11761 TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGAATGTATAAAC TTACATAAAT CTTTTTATTT GTTTATCCCC AAGGCGCGTG TAAAGGGGCT11821 AAAGTGCCAC CTGACGTCTTTCACGGTG GACTGCAG
全文摘要
本發(fā)明涉及一種用關(guān)中奶山羊酪蛋白基因啟動子區(qū)域構(gòu)建的表達(dá)載體。特征是載體DNA序列中包含關(guān)中奶山羊乳腺組織中β－酪蛋白基因的啟動子及其周圍的調(diào)控序列以及β－酪蛋白基因的第一內(nèi)含子、第一外顯子和第二外顯子及其信號肽6.5kb；將這段6.5kb長的序列插入商售的質(zhì)粒載體中，構(gòu)建得到含關(guān)中奶山羊β－酪蛋白基因啟動子區(qū)域序列的重組質(zhì)粒，DNA序列全長為6.5kb加上商售的質(zhì)粒載體序列；實驗結(jié)果充分說明所克隆的6.5kb DNA序列具有啟動子和增強(qiáng)子活性。用本啟動子構(gòu)建的表達(dá)載體能夠用于建立轉(zhuǎn)基因山羊乳腺生物反應(yīng)器，使用基因工程手段所構(gòu)建的目的蛋白特異性地在山羊乳腺中表達(dá)、并分泌到山羊乳汁中，以供制備生產(chǎn)目的蛋白。
文檔編號C12N15/63GK1661018SQ20041007316
公開日2005年8月31日 申請日期2004年10月11日 優(yōu)先權(quán)日2004年10月11日
發(fā)明者陳蘇民, 梁克明, 董德文 申請人:中國人民解放軍第四軍醫(yī)大學(xué)