LOCUS       Exported                9268 bp DNA     circular SYN 02-SEP-2024
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 9268)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9268)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9268)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9268
                     /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(4930..5742)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6121..6869
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7013..7153
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7339..7927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8101..8958)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8959..9063)
                     /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 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 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 agtgagacgt 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 cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat
     4681 accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag
     4741 ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg
     4801 cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac
     4861 aaagccgccg tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa
     4921 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4981 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5041 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5101 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     5161 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
     5221 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     5281 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     5341 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     5401 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     5461 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     5521 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     5581 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     5641 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     5701 tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     5761 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     5821 acatcagaga ttttgagaca caacgtggct ttcccccccc cccctgcagg tcggcatcac
     5881 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     5941 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     6001 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     6061 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     6121 tcgagtatct atgattggaa gtatgggaat ggtgataccc gcattcttca gtgtcttgag
     6181 gtctcctatc agattatgcc caactaaagc aaccggagga ggagatttca tggtaaattt
     6241 ctctgacttt tggtcatcag tagactcgaa ctgtgagact atctcggtta tgacagcaga
     6301 aatgtccttc ttggagacag taaatgaagt cccaccaata aagaaatcct tgttatcagg
     6361 aacaaacttc ttgtttcgaa ctttttcggt gccttgaact ataaaatgta gagtggatat
     6421 gtcgggtagg aatggagcgg gcaaatgctt accttctgga ccttcaagag gtatgtaggg
     6481 tttgtagata ctgatgccaa cttcagtgac aacgttgcta tttcgttcaa accattccga
     6541 atccagagaa atcaaagttg tttgtctact attgatccaa gccagtgcgg tcttgaaact
     6601 gacaatagtg tgctcgtgtt ttgaggtcat ctttgtatga ataaatctag tctttgatct
     6661 aaataatctt gacgagccag acgataatac caatctaaac tctttaaacg ttaaaggaca
     6721 agtatgtctg cctgtattaa accccaaatc agctcgtagt ctgatcctca tcaacttgag
     6781 gggcactatc ttgttttaga gaaatttgcg gagatgcgat atcgagaaaa aggtacgctg
     6841 attttaaacg tgaaatttat ctcaagatct gctgcctcgc gcgtttcggt gatgacggtg
     6901 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     6961 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     7021 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     7081 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     7141 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     7201 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     7261 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     7321 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     7381 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     7441 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     7501 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     7561 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     7621 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     7681 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     7741 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     7801 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     7861 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     7921 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     7981 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     8041 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     8101 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     8161 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     8221 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     8281 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     8341 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     8401 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     8461 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     8521 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     8581 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     8641 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     8701 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     8761 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     8821 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     8881 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     8941 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     9001 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     9061 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     9121 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattaa ttctcatgtt
     9181 tgacagctta tcatcgataa gctgactcat gttggtattg tgaaatagac gcagatcggg
     9241 aacactgaaa aataacagtt attattcg
//
