LOCUS       pPIC9K.        9276 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Pichia pastoris multi-copy integrating vector for methanol-inducible
            expression of a secreted protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPIC9K.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9276)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9276)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             949..1215
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
     misc_feature    1216..1241
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     terminator      1321..1567
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1980..4514)
                     /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(4931..5743)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6122..6878
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7021..7161
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7347..7935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8109..8966)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8967..9071)
                     /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 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 gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca
     4681 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga
     4741 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct
     4801 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa
     4861 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca
     4921 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     4981 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     5041 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     5101 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     5161 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     5221 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     5281 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     5341 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     5401 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     5461 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     5521 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     5581 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     5641 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     5701 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     5761 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     5821 aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca
     5881 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     5941 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     6001 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     6061 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     6121 gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga
     6181 ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt
     6241 tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag
     6301 aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag
     6361 gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata
     6421 tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg
     6481 gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg
     6541 aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac
     6601 tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc
     6661 taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt
     6721 aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat
     6781 caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa
     6841 ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga
     6901 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     6961 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     7021 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     7081 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     7141 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     7201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     7261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     7321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     7381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     7441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     7501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
     7561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     7621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     7681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     7741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     7801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     7861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     7921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     7981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     8041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     8101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     8161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     8221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     8281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     8341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     8401 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     8461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     8521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     8581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     8641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     8701 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     8761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     8821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     8881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     8941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     9001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     9061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     9121 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt
     9181 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc
     9241 agatcgggaa cactgaaaaa taacagttat tattcg
//
