LOCUS       pPIC3.5K.        9004 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Pichia pastoris multi-copy integrating vector for methanol-inducible
            intracellular expression of a protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPIC3.5K.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9004)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9004)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     misc_feature    938..969
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     terminator      1049..1295
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1708..4242)
                     /codon_start=1
                     /gene="Pichia pastoris HIS4"
                     /product="multifunctional enzyme, required for histidine 
                     biosynthesis"
                     /label=Pichia pastoris HIS4
                     /note="PpHIS4"
                     /note="auxotrophic marker for Pichia pastoris"
                     /translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
                     LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
                     VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
                     DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
                     PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
                     TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
                     IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
                     KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
                     LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
                     GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
                     VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
                     LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
                     AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
                     QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
                     HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
     CDS             complement(4659..5471)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    5850..6606
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    6749..6889
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7075..7663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7837..8694)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8695..8799)
                     /label=AmpR promoter
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 tcgtggccga cctgcagggg gggggggggc gctgaggtct
     4381 gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca
     4441 gaaagtgagg gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt
     4501 gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt
     4561 caactcagca aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg
     4621 ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     4681 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     4741 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     4801 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     4861 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     4921 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     4981 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     5041 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     5101 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     5161 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     5221 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     5281 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     5341 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     5401 tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga
     5461 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     5521 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     5581 tccccccccc ccctgcaggt cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     5641 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     5701 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     5761 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     5821 ctaatgcagg agtcgcataa gggagagcgt cgagtatcta tgattggaag tatgggaatg
     5881 gtgatacccg cattcttcag tgtcttgagg tctcctatca gattatgccc aactaaagca
     5941 accggaggag gagatttcat ggtaaatttc tctgactttt ggtcatcagt agactcgaac
     6001 tgtgagacta tctcggttat gacagcagaa atgtccttct tggagacagt aaatgaagtc
     6061 ccaccaataa agaaatcctt gttatcagga acaaacttct tgtttcgaac tttttcggtg
     6121 ccttgaacta taaaatgtag agtggatatg tcgggtagga atggagcggg caaatgctta
     6181 ccttctggac cttcaagagg tatgtagggt ttgtagatac tgatgccaac ttcagtgaca
     6241 acgttgctat ttcgttcaaa ccattccgaa tccagagaaa tcaaagttgt ttgtctacta
     6301 ttgatccaag ccagtgcggt cttgaaactg acaatagtgt gctcgtgttt tgaggtcatc
     6361 tttgtatgaa taaatctagt ctttgatcta aataatcttg acgagccaag gcgataaata
     6421 cccaaatcta aaactctttt aaaacgttaa aaggacaagt atgtctgcct gtattaaacc
     6481 ccaaatcagc tcgtagtctg atcctcatca acttgagggg cactatcttg ttttagagaa
     6541 atttgcggag atgcgatatc gagaaaaagg tacgctgatt ttaaacgtga aatttatctc
     6601 aagatctctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     6661 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     6721 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     6781 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     6841 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     6901 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6961 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     7021 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     7081 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     7141 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     7201 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     7261 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     7321 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7381 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7441 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7501 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7561 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7621 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7681 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7741 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7801 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7861 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7921 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7981 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     8041 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     8101 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt
     8161 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     8221 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     8281 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     8341 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8401 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa
     8461 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8521 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8581 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8641 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8701 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8761 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8821 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     8881 gaggcccttt cgtcttcaag aattaattct catgtttgac agcttatcat cgataagctg
     8941 actcatgttg gtattgtgaa atagacgcag atcgggaaca ctgaaaaata acagttatta
     9001 ttcg
//
