pPIC9

pPIC9

pPIC9载体基本信息

pPIC9载体质粒图谱和多克隆位点信息

pPIC9载体的详细信息如下：

• pPIC9载体大小8,023 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, SnaB I, EcoR I, Avr II, Not I.

• 利用alpha因子分泌信号肽，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)。

pPIC9载体序列

LOCUS       pPIC9           8023 bp ds-DNA    circular SYN
DEFINITION  Pichia pastoris HIS4 vector for methanol-inducible expression of a
            secreted protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPIC9
SOURCE      synthetic DNA construct
ORGANISM  synthetic DNA construct
COMMENT     This file is created by Vector NTI
     source          1..8023
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        2..937
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 promoter"
                     /note="inducible promoter, regulated by methanol"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             949..1215
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae
                     alpha-factor"
                     /note="alpha-factor secretion signal"
                     /note="Cleavage by the Kex2 protease occurs after the
                     dibasic KR sequence. The EA dipeptides are then removed by
                     dipeptidyl aminopeptidase A."
                     /note="This feature has 3 segments:
                     ???1:??949?..?1005?/?#993366?/?presequence
                     ???2:?1006?..?1203?/?#993366?/?pro?region
                     ???3:?1204?..?1215?/?#993366
                     Cleavage sites after bases 1005, 1203"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     misc_feature    1192..1241
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     terminator      1321..1567
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     CDS             complement(1980..4514)
                     /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    4869..5625
                     /note="AOX1 3' fragment"
                     /note="region downstream of Pichia pastoris AOX1 gene"
                     /note="color: #ffffff"
     rep_origin      complement(6094..6682)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6853..7713)
                     /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:?6853?..?7644?/?#ccffcc
                     ???2:?7645?..?7713?/?#ccffcc?/?signal?sequence
                     Cleavage site after base 7644"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7714..7818)
                     /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 gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct
      961 tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact
     1021 acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta
     1081 gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg
     1141 tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa
     1201 agagaggctg aagcttacgt agaattccct agggcggccg cgaattaatt cgccttagac
     1261 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa
     1321 tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt
     1381 ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc
     1441 ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa
     1501 tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta
     1561 agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt
     1621 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc
     1681 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc
     1741 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc
     1801 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg
     1861 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg
     1921 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat
     1981 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct
     2041 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg
     2101 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca
     2161 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga
     2221 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca
     2281 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc
     2341 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc
     2401 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct
     2461 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga
     2521 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa
     2581 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg
     2641 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct
     2701 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca
     2761 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga
     2821 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa
     2881 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt
     2941 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga
     3001 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct
     3061 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact
     3121 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg
     3181 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca
     3241 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct
     3301 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc
     3361 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc
     3421 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca
     3481 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta
     3541 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga
     3601 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca
     3661 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc
     3721 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta
     3781 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata
     3841 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca
     3901 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt
     3961 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga
     4021 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg
     4081 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc
     4141 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca
     4201 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc
     4261 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg
     4321 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc
     4381 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg
     4441 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc
     4501 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg
     4561 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc
     4621 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     4681 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     4741 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     4801 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     4861 ggagagcgtc gagtatctat gattggaagt atgggaatgg tgatacccgc attcttcagt
     4921 gtcttgaggt ctcctatcag attatgccca actaaagcaa ccggaggagg agatttcatg
     4981 gtaaatttct ctgacttttg gtcatcagta gactcgaact gtgagactat ctcggttatg
     5041 acagcagaaa tgtccttctt ggagacagta aatgaagtcc caccaataaa gaaatccttg
     5101 ttatcaggaa caaacttctt gtttcgaact ttttcggtgc cttgaactat aaaatgtaga
     5161 gtggatatgt cgggtaggaa tggagcgggc aaatgcttac cttctggacc ttcaagaggt
     5221 atgtagggtt tgtagatact gatgccaact tcagtgacaa cgttgctatt tcgttcaaac
     5281 cattccgaat ccagagaaat caaagttgtt tgtctactat tgatccaagc cagtgcggtc
     5341 ttgaaactga caatagtgtg ctcgtgtttt gaggtcatct ttgtatgaat aaatctagtc
     5401 tttgatctaa ataatcttga cgagccaagg cgataaatac ccaaatctaa aactctttta
     5461 aaacgttaaa aggacaagta tgtctgcctg tattaaaccc caaatcagct cgtagtctga
     5521 tcctcatcaa cttgaggggc actatcttgt tttagagaaa tttgcggaga tgcgatatcg
     5581 agaaaaaggt acgctgattt taaacgtgaa atttatctca agatctctgc ctcgcgcgtt
     5641 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     5701 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     5761 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     5821 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     5881 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     5941 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6001 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6061 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6121 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6181 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6241 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     6301 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6361 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6421 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6481 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     6541 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6601 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6661 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6721 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6781 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6841 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6901 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6961 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7021 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7081 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7141 tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg
     7201 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7261 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7321 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7381 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7441 gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac
     7501 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7561 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7621 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7681 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7741 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7801 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7861 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga
     7921 attaattctc atgtttgaca gcttatcatc gataagctga ctcatgttgg tattgtgaaa
     7981 tagacgcaga tcgggaacac tgaaaaataa cagttattat tcg
//

pPIC9其他酵母表达载体：