pHIL-S1
pHIL-S1
pHIL-S1
编号 | 载体名称 |
北京华越洋生物VECT3080 | pHIL-S1 |
pHIL-S1载体基本信息
载体名称: | pHIL-S1, pHIL S1 |
---|---|
质粒类型: | 毕赤酵母蛋白表达载体 |
表达水平: | 高拷贝 |
启动子: | AOX1 |
克隆方法: | 多克隆位点,限制性内切酶 |
载体大小: | 8260 bp |
5' 测序引物及序列: | 5´ AOX1:5´-GACTGGTTCCAATTGACAAGC-3´; |
3' 测序引物及序列: | 3´ AOX1:5´-GCAAATGGCATTCTGACATCC-3´ |
载体标签: | N-PHO1分泌信号肽 |
载体抗性: | Ampicillin |
筛选标记: | HIS4 |
备注: | 利用PHO1信号肽,分泌表达蛋白。 |
稳定性: | 稳定 Stable |
组成型: | 组成型 Constitutive |
病毒/非病毒: | 非病毒 |
pHIL-S1载体质粒图谱和多克隆位点信息
pHIL-S1载体简介
pHIL-S1载体的详细信息如下:
• pHIL-S1载体大小8,260 bp,是融合表达载体。
• 载体构建过程中,可供使用的单一限制性内切酶位点为Xho I, EcoR I, Sma I, BamH I.
• 利用PHO1分泌信号肽,分泌表达蛋白基因。
• 在载体构建过程中,你的基因必须保证与信号肽的起始密码子的读码框一致。
• 毕赤酵母中利用HIS4进行筛选。
• 为了将基因插入GS115或KM71的AOX区, 使用SacI限制性内切酶线性化质粒(GS115中产生His+Mut+基因型,KM71中产生His+ MutS基因型)
•为了将基因插入GS115或KM71的His4区, 使用Sal I或者Stu I限制性内切酶线性化质粒(GS115中产生His+ Mut+基因型,KM71中产生His+ MutS基因型)
•将基因插入GS115的AOX1区域,需要使用Bgl II线性化质粒 (产生His+MutS基因型)。
pHIL-S1载体序列
LOCUS pHIL-S1 8260 bp ds-DNA circular SYN 05-二月-2013 DEFINITION Pichia pastoris HIS4 vector for methanol-inducible expression of a secreted protein. ACCESSION . VERSION . KEYWORDS pHIL-S1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct COMMENT This file is created by Vector NTI FEATURES Location/Qualifiers source 1..8260 /organism="synthetic DNA construct" /lab_host="Pichia pastoris" /mol_type="other DNA" promoter 2..941 /gene="Pichia pastoris AOX1" /note="AOX1 promoter" /note="inducible promoter, regulated by methanol" /note="color: #ffffff; direction: RIGHT" CDS 942..1007 /codon_start=1 /product="signal sequence from a secreted acid phosphatase of Pichia pastoris" /note="PHO1 signal sequence" /note="synthetic gene fragment" /note="color: #993366 Cleavage site after base 986" /translation="MFSPILSLEIILALATLQSVFA" misc_feature 1006..1026 /note="MCS" /note="multiple cloning site" /note="color: #99ccff" terminator 1093..1339 /gene="Pichia pastoris AOX1" /note="AOX1 terminator" /note="transcription terminator for AOX1" /note="color: #ffffff" CDS complement(1752..4286) /codon_start=1 /gene="Pichia pastoris HIS4" /locus_tag=" " /product="multifunctional enzyme, required for histidine biosynthesis" /note="PpHIS4" /note="auxotrophic marker for Pichia pastoris" /note="color: #ff7f50" /protein_id=" " /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI" misc_feature 4641..5397 /note="AOX1 3' fragment" /note="region downstream of Pichia pastoris AOX1 gene" /note="color: #ffffff" rep_origin complement(5866..6454) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" rep_origin 6565..7020 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /note="color: #ffff00" CDS complement(7062..7922) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: ???1:?7062?..?7853?/?#ccffcc ???2:?7854?..?7922?/?#ccffcc?/?signal?sequence Cleavage site after base 7853" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7923..8027) /gene="bla" /note="AmpR promoter" /note="color: #ffffff; direction: LEFT" ORIGIN 1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggtcat gtttgtttat 301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga 361 gtgtggggtc aaatagtttc atgttcccaa atggcccaaa actgacagtt taaacgctgt 421 cttggaacct aatatgacaa aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt 481 gaaatgctaa cggccagttg gtcaaaaaga aacttccaaa agtcgccata ccgtttgtct 541 tgtttggtat tgattgacga atgctcaaaa ataatctcat taatgcttag cgcagtctct 601 ctatcgcttc tgaacccggt ggcacctgtg ccgaaacgca aatggggaaa caacccgctt 661 tttggatgat tatgcattgt cctccacatt gtatgcttcc aagattctgg tgggaatact 721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacaggcaa 781 tatataaaca gaaggaagct gccctgtctt aaaccttttt ttttatcatc attattagct 841 tactttcata attgcgactg gttccaattg acaagctttt gattttaacg acttttaacg 901 acaacttgag aagatcaaaa aacaactaat tattcgaaac gatgttctct ccaattttgt 961 ccttggaaat tattttagct ttggctactt tgcaatctgt cttcgctcga gaattccccg 1021 ggatccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt 1081 gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt 1141 tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct 1201 tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg 1261 ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag 1321 tacagaagat taagtgagaa gttcgtttgt gcaagcttat cgataagctt taatgcggta 1381 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc 1441 tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg 1501 tactgccggg cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg 1561 tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt 1621 ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact 1681 acgcgatcat ggcgaccaca cccgtcctgt ggatctatcg aatctaaatg taagttaaaa 1741 tctctaaata attaaataag tcccagtttc tccatacgaa ccttaacagc attgcggtga 1801 gcatctagac cttcaacagc agccagatcc atcactgctt ggccaatatg tttcagtccc 1861 tcaggagtta cgtcttgtga agtgatgaac ttctggaagg ttgcagtgtt aactccgctg 1921 tattgacggg catatccgta cgttggcaaa gtgtggttgg taccggagga gtaatctcca 1981 caactctctg gagagtaggc accaacaaac acagatccag cgtgttgtac ttgatcaaca 2041 taagaagaag cattctcgat ttgcaggatc aagtgttcag gagcgtactg attggacatt 2101 tccaaagcct gctcgtaggt tgcaaccgat agggttgtag agtgtgcaat acacttgcgt 2161 acaatttcaa cccttggcaa ctgcacagct tggttgtgaa cagcatcttc aattctggca 2221 agctccttgt ctgtcatatc gacagccaac agaatcacct gggaatcaat accatgttca 2281 gcttgagaca gaaggtctga ggcaacgaaa tctggatcag cgtatttatc agcaataact 2341 agaacttcag aaggcccagc aggcatgtca atactacaca gggctgatgt gtcattttga 2401 accatcatct tggcagcagt aacgaactgg tttcctggac caaatatttt gtcacactta 2461 ggaacagttt ctgttccgta agccatagca gctactgcct gggcgcctcc tgctagcacg 2521 atacacttag caccaacctt gtgggcaacg tagatgactt ctggggtaag ggtaccatcc 2581 ttcttaggtg gagatgcaaa aacaatttct ttgcaaccag caactttggc aggaacaccc 2641 agcatcaggg aagtggaagg cagaattgcg gttccaccag gaatatagag gccaactttc 2701 tcaataggtc ttgcaaaacg agagcagact acaccagggc aagtctcaac ttgcaacgtc 2761 tccgttagtt gagcttcatg gaatttcctg acgttatcta tagagagatc aatggctctc 2821 ttaacgttat ctggcaattg cataagttcc tctgggaaag gagcttctaa cacaggtgtc 2881 ttcaaagcga ctccatcaaa cttggcagtt agttctaaaa gggctttgtc accattttga 2941 cgaacattgt cgacaattgg tttgactaat tccataatct gttccgtttt ctggatagga 3001 cgacgaaggg catcttcaat ttcttgtgag gaggccttag aaacgtcaat tttgcacaat 3061 tcaatacgac cttcagaagg gacttcttta ggtttggatt cttctttagg ttgttccttg 3121 gtgtatcctg gcttggcatc tcctttcctt ctagtgacct ttagggactt catatccagg 3181 tttctctcca cctcgtccaa cgtcacaccg tacttggcac atctaactaa tgcaaaataa 3241 aataagtcag cacattccca ggctatatct tccttggatt tagcttctgc aagttcatca 3301 gcttcctccc taattttagc gttcaacaaa acttcgtcgt caaataaccg tttggtataa 3361 gaaccttctg gagcattgct cttacgatcc cacaaggtgg cttccatggc tctaagaccc 3421 tttgattggc caaaacagga agtgcgttcc aagtgacaga aaccaacacc tgtttgttca 3481 accacaaatt tcaagcagtc tccatcacaa tccaattcga tacccagcaa cttttgagtt 3541 gctccagatg tagcaccttt ataccacaaa ccgtgacgac gagattggta gactccagtt 3601 tgtgtcctta tagcctccgg aatagacttt ttggacgagt acaccaggcc caacgagtaa 3661 ttagaagagt cagccaccaa agtagtgaat agaccatcgg ggcggtcagt agtcaaagac 3721 gccaacaaaa tttcactgac agggaacttt ttgacatctt cagaaagttc gtattcagta 3781 gtcaattgcc gagcatcaat aatggggatt ataccagaag caacagtgga agtcacatct 3841 accaactttg cggtctcaga aaaagcataa acagttctac taccgccatt agtgaaactt 3901 ttcaaatcgc ccagtggaga agaaaaaggc acagcgatac tagcattagc gggcaaggat 3961 gcaactttat caaccagggt cctatagata accctagcgc ctgggatcat cctttggaca 4021 actctttctg ccaaatctag gtccaaaatc acttcattga taccattatt gtacaacttg 4081 agcaagttgt cgatcagctc ctcaaattgg tcctctgtaa cggatgactc aacttgcaca 4141 ttaacttgaa gctcagtcga ttgagtgaac ttgatcaggt tgtgcagctg gtcagcagca 4201 tagggaaaca cggcttttcc taccaaactc aaggaattat caaactctgc aacacttgcg 4261 tatgcaggta gcaagggaaa tgtcatactt gaagtcggac agtgagtgta gtcttgagaa 4321 attctgaagc cgtattttta ttatcagtga gtcagtcatc aggagatcct ctacgccgga 4381 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac 4441 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg 4501 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca 4561 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag 4621 gagtcgcata agggagagcg tcgagtatct atgattggaa gtatgggaat ggtgataccc 4681 gcattcttca gtgtcttgag gtctcctatc agattatgcc caactaaagc aaccggagga 4741 ggagatttca tggtaaattt ctctgacttt tggtcatcag tagactcgaa ctgtgagact 4801 atctcggtta tgacagcaga aatgtccttc ttggagacag taaatgaagt cccaccaata 4861 aagaaatcct tgttatcagg aacaaacttc ttgtttcgaa ctttttcggt gccttgaact 4921 ataaaatgta gagtggatat gtcgggtagg aatggagcgg gcaaatgctt accttctgga 4981 ccttcaagag gtatgtaggg tttgtagata ctgatgccaa cttcagtgac aacgttgcta 5041 tttcgttcaa accattccga atccagagaa atcaaagttg tttgtctact attgatccaa 5101 gccagtgcgg tcttgaaact gacaatagtg tgctcgtgtt ttgaggtcat ctttgtatga 5161 ataaatctag tctttgatct aaataatctt gacgagccaa ggcgataaat acccaaatct 5221 aaaactcttt taaaacgtta aaaggacaag tatgtctgcc tgtattaaac cccaaatcag 5281 ctcgtagtct gatcctcatc aacttgaggg gcactatctt gttttagaga aatttgcgga 5341 gatgcgatat cgagaaaaag gtacgctgat tttaaacgtg aaatttatct caagatctct 5401 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 5461 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 5521 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 5581 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 5641 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 5701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 5821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 5881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 5941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6061 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6301 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6541 aaggatcttc acctagatcc ttttacgcgc cctgtagcgg cgcattaagc gcggcgggtg 6601 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 6661 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 6721 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 6781 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 6841 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 6901 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 6961 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca 7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca 7681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 7741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 7801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 7861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 7921 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7981 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 8041 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 8101 cgtatcacga ggccctttcg tcttcaagat ttctcatgtt tgacagctta tcatcgaatt 8161 aattctcatg tttgacagct tatcatcgat aagctgactc atgttggtat tgtgaaatag 8221 acgcagatcg ggaacactga aaaataacag ttattattcg //
pHIL-S1其他酵母表达载体: