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基本信息

質(zhì)粒圖譜和多克隆位點(diǎn)信息

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載體描述

泛素(ubiquitin)分子量很小，由 76 aa殘基組成；泛素作為降解信號(hào)分子，可以連接另外一種蛋白質(zhì)的 N端，然后被泛素專一性蛋白酶 (UBPs)識(shí)別，從而導(dǎo)致與泛素相連的蛋白被酶解。泛素可以人為分成兩部分：N端 (Nub)，C端 (Cub)。首先，人為地將泛素 Nub的 3位異亮氨酸突變?yōu)楦拾彼?(NubI 突變?yōu)?NubG)。這樣與 Cub的親合力大大降低，避免了 Cub和 Nub自我結(jié)合或接近的可能性。其次，將Cub部分與人工合成的 LexA-VP16轉(zhuǎn)錄激活因子融合成一個(gè)融合蛋白Cub-LexA-VP16。正常條件下 NubG不與Cub 結(jié)合，UBPs也不能識(shí)別分離的泛素，轉(zhuǎn)錄激活因子也不會(huì)被切下來(lái)。最后，將要檢測(cè)的蛋白質(zhì)分別與NubG和Cub融合，形成 bait融合蛋 (bait-cub-LexA-VP16)和 prey融合蛋白 (prey-NubG)。如果 bait和 prey發(fā)生相互作用，就會(huì)促使 NubG和 Cub的相互接近，被 UBPs識(shí)別，導(dǎo)致 LexA-VP16的解離，進(jìn)入核內(nèi)，從而激活報(bào)告基因的轉(zhuǎn)錄。

膜體系酵母雙雜交體系中根據(jù)誘餌蛋白質(zhì)定位不同，需要選用不同的載體，以便于與互作蛋白相連的泛素NubG和Cub-LexA-VP16可以相互作用，從而被泛素特異性蛋白酶（UBPs）識(shí)別、切割、啟動(dòng)報(bào)告基因表達(dá)。

載體序列

ORIGIN

        1 gccgcggatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag

       61 ctctcatttg gcgagcgttg gttggtggat caagcccacg cgtaggcaat cctcgagcag

      121 atccgccagg cgtgtatata tagcgtggat ggccaggcaa ctttagtgct gacacataca

      181 ggcatatata tatgtgtgcg acgacacatg atcatatggc atgcatgtgc tctgtatgta

      241 tataaaactc ttgttttctt cttttctcta aatattcttt ccttatacat taggaccttt

      301 gcagcataaa ttactatact tctatagaca cacaaacaca aatacacaca ctaatctaga

      361 actagtatgc agattttcgt caagactttg accggtaaaa ccggaacatt ggaagttgaa

      421 tcttccgata ccatcgacaa cgttaagtcg aaaattcaag acaaggaagg aatccctggt

      481 ggtccatacc catacgatgt tccagattac gctggatcca agcagtggta tcaacgcaga

      541 gtggccatta cggcccggga aaaaacatgt cggccgcctc ggcctctcga gaattcgata

      601 tcaagcttat cgataccgtc gacctcgagt catgtaatta gttatgtcac gcttacattc

      661 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta

      721 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat

      781 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct

      841 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat

      901 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac

      961 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat

     1021 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg

     1081 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg

     1141 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca

     1201 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta

     1261 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc

     1321 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg

     1381 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat

     1441 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta

     1501 acgcgaattt taacaaaata ttaacgttta caatttcctg atgcgtattt tctccttacg

     1561 catctgtgcg gtatttcaca ccgcataggc aagtgcacaa acaatactta aataaatact

     1621 actcagtaat aacctatttc ttagcatttt tgacgaaatt tgctattttg ttagagtctt

     1681 ttacaccatt tgtctccaca cctccgctta catcaacacc aataacgcca tttaatctaa

     1741 gcgcatcacc aacattttct ggcgtcagtc caccagctaa cataaaatgt aagctttcgg

     1801 ggctctcttg ccttccaacc cagtcagaaa tcgagttcca atccaaaagt tcacctgtcc

     1861 cacctgcttc tgaatcaaac aagggaataa acgaatgagg tttctgtgaa gctgcactga

     1921 gtagtatgtt gcagtctttt ggaaatacga gtcttttaat aactggcaaa ccgaggaact

     1981 cttggtattc ttgccacgac tcatctccat gcagttggac gatatcaatg ccgtaatcat

     2041 tgaccagagc caaaacatcc tccttaggtt gattacgaaa cacgccaacc aagtatttcg

     2101 gagtgcctga actattttta tatgctttta caagacttga aattttcctt gcaataaccg

     2161 ggtcaattgt tctctttcta ttgggcacac atataatacc cagcaagtca gcatcggaat

     2221 ctagagcaca ttctgcggcc tctgtgctct gcaagccgca aactttcacc aatggaccag

     2281 aactacctgt gaaattaata acagacatac tccaagctgc ctttgtgtgc ttaatcacgt

     2341 atactcacgt gctcaatagt caccaatgcc ctccctcttg gccctctcct tttctttttt

     2401 cgaccgaatt aattcttaat cggcaaaaaa agaaaagctc cggatcaaga ttgtacgtaa

     2461 ggtgacaagc tatttttcaa taaagaatat cttccactac tgccatctgg cgtcataact

     2521 gcaaagtaca catatattac gatgctgtct attaaatgct tcctatatta tatatatagt

     2581 aatgtcgttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc

     2641 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg

     2701 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat

     2761 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca

     2821 tgataataat ggtttcttaa tatgatccaa tatcaaagga aatgatagca ttgaaggatg

     2881 agactaatcc aattgaggag tggcagcata tagaacagct aaagggtagt gctgaaggaa

     2941 gcatacgata ccccgcatgg aatgggataa tatcacagga ggtactagac tacctttcat

     3001 cctacataaa tagacgcata taagtacgca tttaagcata aacacgcact atgccgttct

     3061 tctcatgtat atatatatac aggcaacacg cagatatagg tgcgacgtga acagtgagct

     3121 gtatgtgcgc agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac gctttgaagt

     3181 tcctattccg aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaaaac

     3241 caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct

     3301 actaaaatat tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat

     3361 atctctgtgc tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat

     3421 ctaaactcga cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa

     3481 ttgtagtaag aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct

     3541 atacgtagta tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga

     3601 atcatcaacg ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata

     3661 taatcgggga tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa

     3721 cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc

     3781 ttctaacctt aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca

     3841 caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca

     3901 tttttgtaga acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg

     3961 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg

     4021 ttgcattttt gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg

     4081 cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc

     4141 tcgcgttgca tttttgttct acaaaatgaa gcacagatgc ttcgttcagg tggcactttt

     4201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat

     4261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg

     4321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt

     4381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga

     4441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa

     4501 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt

     4561 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt

     4621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc

     4681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga

     4741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat

     4801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct

     4861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc

     4921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg

     4981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc

     5041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg

     5101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca

     5161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta

     5221 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc

     5281 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa

     5341 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca

     5401 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta

     5461 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc

     5521 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca

     5581 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta

     5641 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag

     5701 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt

     5761 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc

     5821 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac

     5881 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac

     5941 gccagcaacg cggcttttta cggttcctgg ccttttgctg gccttttgct cacatgttct

     6001 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata

     6061 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc

     6121 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg

     6181 acaggtttcc cgactggaaa

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