pAO815

pAO815

载体基本信息

出品公司:	Ybscience
载体名称:	pAO815
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:	7709 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:	/
载体抗性:	Ampicillin
筛选标记:	HIS4
备注:	插入基因是必需包含起始密码子ATG。
稳定性:	稳定 Stable
组成型:	组成型 Constitutive
病毒/非病毒:	非病毒

载体质粒图谱和多克隆位点信息

载体简介

pAO815是一个鉴定多拷贝基因插入到毕赤酵母基因组中的载体。

1. pAO815载体大小为7709 bp。

2. 在多克隆位点区域，拥有1个单一的限制性酶切位点EcoR I。

3.胞内表达。

4.载体构建时，需要包含一个ATG启动子，同时需要加入Kozak序列。 (Cavener and Stuart, 1991; Kozak, 1987; Kozak, 1990)

5. 毕赤酵母中使用HIS4基因筛选。

6. 将基因插入GS115和KM71的HIS4中，需要使用Sal I或者Stu I线性化质粒(GS115产生的基因型为His+ Mut+，KM71酵母产生的基因型为His+ MutS)。

7. 将基因插入GS115的AOX1基因中，也可以使用Bgl II 限制酶线性化质粒，产生的酵母基因型为His+ MutS。

载体序列

LOCUS       pAO815           7709 bp ds-DNA    circular SYN 05-二月-2013
DEFINITION  Pichia pastoris?vector for creating multimers to drive high levels 
            of methanol-inducible protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    pAO815
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     VNTAUTHORNAME|biofeng.com|
COMMENT     The gene of interest can be inserted at the unique EcoRI site.
            
            Multimers of the resulting BglII-BamHI expression cassette can be 
            inserted at the BamHI site.
FEATURES             Location/Qualifiers
     source          1..7709
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 promoter"
                     /note="inducible promoter, regulated by methanol"
                     /note="color: #ffffff; direction: RIGHT"
     terminator      1018..1264
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     CDS             complement(1665..4199)
                     /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    4554..5310
                     /note="AOX1 3' fragment"
                     /note="region downstream of Pichia pastoris AOX1 gene"
                     /note="color: #ffffff"
     rep_origin      complement(5780..6368)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6539..7399)
                     /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:?6539?..?7330?/?#ccffcc
                     ???2:?7331?..?7399?/?#ccffcc?/?signal?sequence
                     Cleavage site after base 7330"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7400..7504)
                     /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 aaagtcggca 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 attcgaaacg aggaattcgc cttagacatg
      961 actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca
     1021 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
     1081 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
     1141 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
     1201 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
     1261 gagacgttcg tttgtgcgga tcctaatgcg gtagtttatc acagttaaat tgctaacgca
     1321 gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt catcctcggc accgtcaccc
     1381 tggatgctgt aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg
     1441 tccattccga cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc
     1501 aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc
     1561 tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc
     1621 tgtggatcta tcgaatctaa atgtaagtta aaatctctaa ataattaaat aagtcccagt
     1681 ttctccatac gaaccttaac agcattgcgg tgagcatcta gaccttcaac agcagccaga
     1741 tccatcactg cttggccaat atgtttcagt ccctcaggag ttacgtcttg tgaagtgatg
     1801 aacttctgga aggttgcagt gttaactccg ctgtattgac gggcatatcc gtacgttggc
     1861 aaagtgtggt tggtaccgga ggagtaatct ccacaactct ctggagagta ggcaccaaca
     1921 aacacagatc cagcgtgttg tacttgatca acataagaag aagcattctc gatttgcagg
     1981 atcaagtgtt caggagcgta ctgattggac atttccaaag cctgctcgta ggttgcaacc
     2041 gatagggttg tagagtgtgc aatacacttg cgtacaattt caacccttgg caactgcaca
     2101 gcttggttgt gaacagcatc ttcaattctg gcaagctcct tgtctgtcat atcgacagcc
     2161 aacagaatca cctgggaatc aataccatgt tcagcttgag acagaaggtc tgaggcaacg
     2221 aaatctggat cagcgtattt atcagcaata actagaactt cagaaggccc agcaggcatg
     2281 tcaatactac acagggctga tgtgtcattt tgaaccatca tcttggcagc agtaacgaac
     2341 tggtttcctg gaccaaatat tttgtcacac ttaggaacag tttctgttcc gtaagccata
     2401 gcagctactg cctgggcgcc tcctgctagc acgatacact tagcaccaac cttgtgggca
     2461 acgtagatga cttctggggt aagggtacca tccttcttag gtggagatgc aaaaacaatt
     2521 tctttgcaac cagcaacttt ggcaggaaca cccagcatca gggaagtgga aggcagaatt
     2581 gcggttccac caggaatata gaggccaact ttctcaatag gtcttgcaaa acgagagcag
     2641 actacaccag ggcaagtctc aacttgcaac gtctccgtta gttgagcttc atggaatttc
     2701 ctgacgttat ctatagagag atcaatggct ctcttaacgt tatctggcaa ttgcataagt
     2761 tcctctggga aaggagcttc taacacaggt gtcttcaaag cgactccatc aaacttggca
     2821 gttagttcta aaagggcttt gtcaccattt tgacgaacat tgtcgacaat tggtttgact
     2881 aattccataa tctgttccgt tttctggata ggacgacgaa gggcatcttc aatttcttgt
     2941 gaggaggcct tagaaacgtc aattttgcac aattcaatac gaccttcaga agggacttct
     3001 ttaggtttgg attcttcttt aggttgttcc ttggtgtatc ctggcttggc atctcctttc
     3061 cttctagtga cctttaggga cttcatatcc aggtttctct ccacctcgtc caacgtcaca
     3121 ccgtacttgg cacatctaac taatgcaaaa taaaataagt cagcacattc ccaggctata
     3181 tcttccttgg atttagcttc tgcaagttca tcagcttcct ccctaatttt agcgttcaac
     3241 aaaacttcgt cgtcaaataa ccgtttggta taagaacctt ctggagcatt gctcttacga
     3301 tcccacaagg tggcttccat ggctctaaga ccctttgatt ggccaaaaca ggaagtgcgt
     3361 tccaagtgac agaaaccaac acctgtttgt tcaaccacaa atttcaagca gtctccatca
     3421 caatccaatt cgatacccag caacttttga gttgctccag atgtagcacc tttataccac
     3481 aaaccgtgac gacgagattg gtagactcca gtttgtgtcc ttatagcctc cggaatagac
     3541 tttttggacg agtacaccag gcccaacgag taattagaag agtcagccac caaagtagtg
     3601 aatagaccat cggggcggtc agtagtcaaa gacgccaaca aaatttcact gacagggaac
     3661 tttttgacat cttcagaaag ttcgtattca gtagtcaatt gccgagcatc aataatgggg
     3721 attataccag aagcaacagt ggaagtcaca tctaccaact ttgcggtctc agaaaaagca
     3781 taaacagttc tactaccgcc attagtgaaa cttttcaaat cgcccagtgg agaagaaaaa
     3841 ggcacagcga tactagcatt agcgggcaag gatgcaactt tatcaaccag ggtcctatag
     3901 ataaccctag cgcctgggat catcctttgg acaactcttt ctgccaaatc taggtccaaa
     3961 atcacttcat tgataccatt attgtacaac ttgagcaagt tgtcgatcag ctcctcaaat
     4021 tggtcctctg taacggatga ctcaacttgc acattaactt gaagctcagt cgattgagtg
     4081 aacttgatca ggttgtgcag ctggtcagca gcatagggaa acacggcttt tcctaccaaa
     4141 ctcaaggaat tatcaaactc tgcaacactt gcgtatgcag gtagcaaggg aaatgtcata
     4201 cttgaagtcg gacagtgagt gtagtcttga gaaattctga agccgtattt ttattatcag
     4261 tgagtcagtc atcaggagat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     4321 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     4381 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     4441 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     4501 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagta
     4561 tctatgattg gaagtatggg aatggtgata cccgcattct tcagtgtctt gaggtctcct
     4621 atcagattat gcccaactaa agcaaccgga ggaggagatt tcatggtaaa tttctctgac
     4681 ttttggtcat cagtagactc gaactgtgag actatctcgg ttatgacagc agaaatgtcc
     4741 ttcttggaga cagtaaatga agtcccacca ataaagaaat ccttgttatc aggaacaaac
     4801 ttcttgtttc gaactttttc ggtgccttga actataaaat gtagagtgga tatgtcgggt
     4861 aggaatggag cgggcaaatg cttaccttct ggaccttcaa gaggtatgta gggtttgtag
     4921 atactgatgc caacttcagt gacaacgttg ctatttcgtt caaaccattc cgaatccaga
     4981 gaaatcaaag ttgtttgtct actattgatc caagccagtg cggtcttgaa actgacaata
     5041 gtgtgctcgt gttttgaggt catctttgta tgaataaatc tagtctttga tctaaataat
     5101 cttgacgagc caaggcgata aatacccaaa tctaaaactc ttttaaaacg ttaaaaggac
     5161 aagtatgtct gcctgtatta aaccccaaat cagctcgtag tctgatcctc atcaacttga
     5221 ggggcactat cttgttttag agaaatttgc ggagatgcga tatcgagaaa aaggtacgct
     5281 gattttaaac gtgaaattta tctcaagatc tgctgcctcg cgcgtttcgg tgatgacggt
     5341 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     5401 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     5461 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     5521 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     5581 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6841 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7141 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7501 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7561 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaatta attctcatgt
     7621 ttgacagctt atcatcgata agctgactca tgttggtatt gtgaaataga cgcagatcgg
     7681 gaacactgaa aaataacagt tattattcg