LOCUS       pAO815        7707 bp DNA     circular SYN 20-JUL-2025
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
REFERENCE   1  (bases 1 to 7707)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7707)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7707)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
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..7707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      1018..1264
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1664..4198)
                     /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"
     misc_feature    4553..5308
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    5452..5592
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5778..6366)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6540..7397)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7398..7502)
                     /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 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 tccaatgcgg tagtttatca cagttaaatt gctaacgcag
     1321 tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc atcctcggca ccgtcaccct
     1381 ggatgctgta ggcataggct tggttatgcc ggtactgccg ggcctcttgc gggatatcgt
     1441 ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg cgttgatgca
     1501 atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc gcccagtcct
     1561 gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca cacccgtcct
     1621 gtggatctat cgaatctaaa tgtaagttaa aatctctaaa taattaaata agtcccagtt
     1681 tctccatacg aaccttaaca gcattgcggt gagcatctag accttcaaca gcagccagat
     1741 ccatcactgc ttggccaata tgtttcagtc cctcaggagt tacgtcttgt gaagtgatga
     1801 acttctggaa ggttgcagtg ttaactccgc tgtattgacg ggcatatccg tacgttggca
     1861 aagtgtggtt ggtaccggag gagtaatctc cacaactctc tggagagtag gcaccaacaa
     1921 acacagatcc agcgtgttgt acttgatcaa cataagaaga agcattctcg atttgcagga
     1981 tcaagtgttc aggagcgtac tgattggaca tttccaaagc ctgctcgtag gttgcaaccg
     2041 atagggttgt agagtgtgca atacacttgc gtacaatttc aacccttggc aactgcacag
     2101 cttggttgtg aacagcatct tcaattctgg caagctcctt gtctgtcata tcgacagcca
     2161 acagaatcac ctgggaatca ataccatgtt cagcttgaga cagaaggtct gaggcaacga
     2221 aatctggatc agcgtattta tcagcaataa ctagaacttc agaaggccca gcaggcatgt
     2281 caatactaca cagggctgat gtgtcatttt gaaccatcat cttggcagca gtaacgaact
     2341 ggtttcctgg accaaatatt ttgtcacact taggaacagt ttctgttccg taagccatag
     2401 cagctactgc ctgggcgcct cctgctagca cgatacactt agcaccaacc ttgtgggcaa
     2461 cgtagatgac ttctggggta agggtaccat ccttcttagg tggagatgca aaaacaattt
     2521 ctttgcaacc agcaactttg gcaggaacac ccagcatcag ggaagtggaa ggcagaattg
     2581 cggttccacc aggaatatag aggccaactt tctcaatagg tcttgcaaaa cgagagcaga
     2641 ctacaccagg gcaagtctca acttgcaacg tctccgttag ttgagcttca tggaatttcc
     2701 tgacgttatc tatagagaga tcaatggctc tcttaacgtt atctggcaat tgcataagtt
     2761 cctctgggaa aggagcttct aacacaggtg tcttcaaagc gactccatca aacttggcag
     2821 ttagttctaa aagggctttg tcaccatttt gacgaacatt gtcgacaatt ggtttgacta
     2881 attccataat ctgttccgtt ttctggatag gacgacgaag ggcatcttca atttcttgtg
     2941 aggaggcctt agaaacgtca attttgcaca attcaatacg accttcagaa gggacttctt
     3001 taggtttgga ttcttcttta ggttgttcct tggtgtatcc tggcttggca tctcctttcc
     3061 ttctagtgac ctttagggac ttcatatcca ggtttctctc cacctcgtcc aacgtcacac
     3121 cgtacttggc acatctaact aatgcaaaat aaaataagtc agcacattcc caggctatat
     3181 cttccttgga tttagcttct gcaagttcat cagcttcctc cctaatttta gcgttcaaca
     3241 aaacttcgtc gtcaaataac cgtttggtat aagaaccttc tggagcattg ctcttacgat
     3301 cccacaaggt ggcttccatg gctctaagac cctttgattg gccaaaacag gaagtgcgtt
     3361 ccaagtgaca gaaaccaaca cctgtttgtt caaccacaaa tttcaagcag tctccatcac
     3421 aatccaattc gatacccagc aacttttgag ttgctccaga tgtagcacct ttataccaca
     3481 aaccgtgacg acgagattgg tagactccag tttgtgtcct tatagcctcc ggaatagact
     3541 ttttggacga gtacaccagg cccaacgagt aattagaaga gtcagccacc aaagtagtga
     3601 atagaccatc ggggcggtca gtagtcaaag acgccaacaa aatttcactg acagggaact
     3661 ttttgacatc ttcagaaagt tcgtattcag tagtcaattg ccgagcatca ataatgggga
     3721 ttataccaga agcaacagtg gaagtcacat ctaccaactt tgcggtctca gaaaaagcat
     3781 aaacagttct actaccgcca ttagtgaaac ttttcaaatc gcccagtgga gaagaaaaag
     3841 gcacagcgat actagcatta gcgggcaagg atgcaacttt atcaaccagg gtcctataga
     3901 taaccctagc gcctgggatc atcctttgga caactctttc tgccaaatct aggtccaaaa
     3961 tcacttcatt gataccatta ttgtacaact tgagcaagtt gtcgatcagc tcctcaaatt
     4021 ggtcctctgt aacggatgac tcaacttgca cattaacttg aagctcagtc gattgagtga
     4081 acttgatcag gttgtgcagc tggtcagcag catagggaaa cacggctttt cctaccaaac
     4141 tcaaggaatt atcaaactct gcaacacttg cgtatgcagg tagcaaggga aatgtcatac
     4201 ttgaagtcgg acagtgagtg tagtcttgag aaattctgaa gccgtatttt tattatcagt
     4261 gagtcagtca tcaggagatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca
     4321 caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc
     4381 acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg
     4441 gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc
     4501 tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagtat
     4561 ctatgattgg aagtatggga atggtgatac ccgcattctt cagtgtcttg aggtctccta
     4621 tcagattatg cccaactaaa gcaaccggag gaggagattt catggtaaat ttctctgact
     4681 tttggtcatc agtagactcg aactgtgaga ctatctcggt tatgacagca gaaatgtcct
     4741 tcttggagac agtaaatgaa gtcccaccaa taaagaaatc cttgttatca ggaacaaact
     4801 tcttgtttcg aactttttcg gtgccttgaa ctataaaatg tagagtggat atgtcgggta
     4861 ggaatggagc gggcaaatgc ttaccttctg gaccttcaag aggtatgtag ggtttgtaga
     4921 tactgatgcc aacttcagtg acaacgttgc tatttcgttc aaaccattcc gaatccagag
     4981 aaatcaaagt tgtttgtcta ctattgatcc aagccagtgc ggtcttgaaa ctgacaatag
     5041 tgtgctcgtg ttttgaggtc atctttgtat gaataaatct agtctttgat ctaaataatc
     5101 ttgacgagcc aaggcgataa atacccaaat ctaaaactct tttaaaacgt taaaggacaa
     5161 gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat caacttgagg
     5221 ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa ggtacgctga
     5281 ttttaaacgt gaaatttatc tcaagatctg ctgcctcgcg cgtttcggtg atgacggtga
     5341 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5401 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     5461 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     5521 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     5581 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5641 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5701 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5761 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5821 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5881 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5941 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6001 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6061 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6121 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6181 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     6241 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6301 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6361 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6421 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6481 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6541 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6601 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6661 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6721 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6781 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6841 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6901 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6961 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7021 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7081 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7141 tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7201 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7261 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7321 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7381 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7441 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7501 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7561 acctataaaa ataggcgtat cacgaggccc tttcgtcttc aagaattaat tctcatgttt
     7621 gacagcttat catcgataag ctgactcatg ttggtattgt gaaatagacg cagatcggga
     7681 acactgaaaa ataacagtta ttattcg
//
