pPIC3.5

pPIC3.5

pPIC3.5载体基本信息

载体质粒图谱和多克隆位点信息

pPIC3.5载体简介

pPIC3.5酵母表达载体详细信息如下:

1.  载体大小为7,751 bp，是非融合蛋白表达载体。

2. 单一限制性内切酶位点有BamH I, SnaB I, EcoR I, Avr II 和Not I.

3. 胞内基因表达载体，不是分泌性蛋白表达载体。

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AOX1中，需要使用Sac 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和KM71的HIS4中，需要使用Sal I或 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)。

8. 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。

pPIC3.5载体序列

LOCUS      pPIC3.5          7751 bp    ds-DNA    circular SYN 05-二月-2013
DEFINITION  Pichia pastoris HIS4 vector for methanol-inducible intracellular
            expression of a protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPIC3.5
COMMENT     This file is created by Vector NTI
FEATURES             Location/Qualifiers
     source          1..7751
                     /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"
     misc_feature    938..969
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     terminator      1049..1295
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     CDS             complement(1708..4242)
                     /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    4597..5353
                     /note="AOX1 3' fragment"
                     /note="region downstream of Pichia pastoris AOX1 gene"
                     /note="color: #ffffff"
     rep_origin      complement(5822..6410)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6581..7441)
                     /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:?6581?..?7372?/?#ccffcc
                     ???2:?7373?..?7441?/?#ccffcc?/?signal?sequence
                     Cleavage site after base 7372"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7442..7546)
                     /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 tcctacgtag aattccctag
      961 ggcggccgcg aattaattcg ccttagacat gactgttcct cagttcaagt tgggcactta
     1021 cgagaagacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag
     1081 agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt
     1141 ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg
     1201 aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc
     1261 ccactcctct tcagagtaca gaagattaag tgagaagttc gtttgtgcaa gcttatcgat
     1321 aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg
     1381 aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata
     1441 ggcttggtta tgccggtact gccgggcctc ttgcgggata tcgtccattc cgacagcatc
     1501 gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct atgcgcaccc
     1561 gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc ttcgctactt
     1621 ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtggat ctatcgaatc
     1681 taaatgtaag ttaaaatctc taaataatta aataagtccc agtttctcca tacgaacctt
     1741 aacagcattg cggtgagcat ctagaccttc aacagcagcc agatccatca ctgcttggcc
     1801 aatatgtttc agtccctcag gagttacgtc ttgtgaagtg atgaacttct ggaaggttgc
     1861 agtgttaact ccgctgtatt gacgggcata tccgtacgtt ggcaaagtgt ggttggtacc
     1921 ggaggagtaa tctccacaac tctctggaga gtaggcacca acaaacacag atccagcgtg
     1981 ttgtacttga tcaacataag aagaagcatt ctcgatttgc aggatcaagt gttcaggagc
     2041 gtactgattg gacatttcca aagcctgctc gtaggttgca accgataggg ttgtagagtg
     2101 tgcaatacac ttgcgtacaa tttcaaccct tggcaactgc acagcttggt tgtgaacagc
     2161 atcttcaatt ctggcaagct ccttgtctgt catatcgaca gccaacagaa tcacctggga
     2221 atcaatacca tgttcagctt gagacagaag gtctgaggca acgaaatctg gatcagcgta
     2281 tttatcagca ataactagaa cttcagaagg cccagcaggc atgtcaatac tacacagggc
     2341 tgatgtgtca ttttgaacca tcatcttggc agcagtaacg aactggtttc ctggaccaaa
     2401 tattttgtca cacttaggaa cagtttctgt tccgtaagcc atagcagcta ctgcctgggc
     2461 gcctcctgct agcacgatac acttagcacc aaccttgtgg gcaacgtaga tgacttctgg
     2521 ggtaagggta ccatccttct taggtggaga tgcaaaaaca atttctttgc aaccagcaac
     2581 tttggcagga acacccagca tcagggaagt ggaaggcaga attgcggttc caccaggaat
     2641 atagaggcca actttctcaa taggtcttgc aaaacgagag cagactacac cagggcaagt
     2701 ctcaacttgc aacgtctccg ttagttgagc ttcatggaat ttcctgacgt tatctataga
     2761 gagatcaatg gctctcttaa cgttatctgg caattgcata agttcctctg ggaaaggagc
     2821 ttctaacaca ggtgtcttca aagcgactcc atcaaacttg gcagttagtt ctaaaagggc
     2881 tttgtcacca ttttgacgaa cattgtcgac aattggtttg actaattcca taatctgttc
     2941 cgttttctgg ataggacgac gaagggcatc ttcaatttct tgtgaggagg ccttagaaac
     3001 gtcaattttg cacaattcaa tacgaccttc agaagggact tctttaggtt tggattcttc
     3061 tttaggttgt tccttggtgt atcctggctt ggcatctcct ttccttctag tgacctttag
     3121 ggacttcata tccaggtttc tctccacctc gtccaacgtc acaccgtact tggcacatct
     3181 aactaatgca aaataaaata agtcagcaca ttcccaggct atatcttcct tggatttagc
     3241 ttctgcaagt tcatcagctt cctccctaat tttagcgttc aacaaaactt cgtcgtcaaa
     3301 taaccgtttg gtataagaac cttctggagc attgctctta cgatcccaca aggtggcttc
     3361 catggctcta agaccctttg attggccaaa acaggaagtg cgttccaagt gacagaaacc
     3421 aacacctgtt tgttcaacca caaatttcaa gcagtctcca tcacaatcca attcgatacc
     3481 cagcaacttt tgagttgctc cagatgtagc acctttatac cacaaaccgt gacgacgaga
     3541 ttggtagact ccagtttgtg tccttatagc ctccggaata gactttttgg acgagtacac
     3601 caggcccaac gagtaattag aagagtcagc caccaaagta gtgaatagac catcggggcg
     3661 gtcagtagtc aaagacgcca acaaaatttc actgacaggg aactttttga catcttcaga
     3721 aagttcgtat tcagtagtca attgccgagc atcaataatg gggattatac cagaagcaac
     3781 agtggaagtc acatctacca actttgcggt ctcagaaaaa gcataaacag ttctactacc
     3841 gccattagtg aaacttttca aatcgcccag tggagaagaa aaaggcacag cgatactagc
     3901 attagcgggc aaggatgcaa ctttatcaac cagggtccta tagataaccc tagcgcctgg
     3961 gatcatcctt tggacaactc tttctgccaa atctaggtcc aaaatcactt cattgatacc
     4021 attattgtac aacttgagca agttgtcgat cagctcctca aattggtcct ctgtaacgga
     4081 tgactcaact tgcacattaa cttgaagctc agtcgattga gtgaacttga tcaggttgtg
     4141 cagctggtca gcagcatagg gaaacacggc ttttcctacc aaactcaagg aattatcaaa
     4201 ctctgcaaca cttgcgtatg caggtagcaa gggaaatgtc atacttgaag tcggacagtg
     4261 agtgtagtct tgagaaattc tgaagccgta tttttattat cagtgagtca gtcatcagga
     4321 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     4381 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     4441 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     4501 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     4561 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gtatctatga ttggaagtat
     4621 gggaatggtg atacccgcat tcttcagtgt cttgaggtct cctatcagat tatgcccaac
     4681 taaagcaacc ggaggaggag atttcatggt aaatttctct gacttttggt catcagtaga
     4741 ctcgaactgt gagactatct cggttatgac agcagaaatg tccttcttgg agacagtaaa
     4801 tgaagtccca ccaataaaga aatccttgtt atcaggaaca aacttcttgt ttcgaacttt
     4861 ttcggtgcct tgaactataa aatgtagagt ggatatgtcg ggtaggaatg gagcgggcaa
     4921 atgcttacct tctggacctt caagaggtat gtagggtttg tagatactga tgccaacttc
     4981 agtgacaacg ttgctatttc gttcaaacca ttccgaatcc agagaaatca aagttgtttg
     5041 tctactattg atccaagcca gtgcggtctt gaaactgaca atagtgtgct cgtgttttga
     5101 ggtcatcttt gtatgaataa atctagtctt tgatctaaat aatcttgacg agccaaggcg
     5161 ataaataccc aaatctaaaa ctcttttaaa acgttaaaag gacaagtatg tctgcctgta
     5221 ttaaacccca aatcagctcg tagtctgatc ctcatcaact tgaggggcac tatcttgttt
     5281 tagagaaatt tgcggagatg cgatatcgag aaaaaggtac gctgatttta aacgtgaaat
     5341 ttatctcaag atctctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5401 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     5461 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     5521 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     5581 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     5641 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5701 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5761 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5821 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5881 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5941 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6001 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6061 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6121 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6181 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6241 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     6301 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6361 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6421 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6481 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6541 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6601 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6661 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6721 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6781 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6841 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     6901 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6961 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7021 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7081 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7141 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     7201 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7261 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7321 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7381 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7441 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7501 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7561 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     7621 gtatcacgag gccctttcgt cttcaagaat taattctcat gtttgacagc ttatcatcga
     7681 taagctgact catgttggta ttgtgaaata gacgcagatc gggaacactg aaaaataaca
     7741 gttattattc g
//

pPIC3.5其他酵母表达载体：