LOCUS       pHIL-S1.        8260 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Pichia pastoris HIS4 vector for methanol-inducible expression of a 
            secreted protein.
ACCESSION   .
VERSION     .
KEYWORDS    pHIL-S1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8260)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8260)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..941
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     sig_peptide     942..1007
                     /label=PHO1 signal sequence
                     /note="signal sequence from a secreted acid phosphatase of 
                     Pichia pastoris"
     misc_feature    1006..1026
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     terminator      1093..1339
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1752..4286)
                     /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    4641..5397
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    5540..5680
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(5866..6454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      6565..7020
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(7065..7922)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7923..8027)
                     /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 gcgaggtcat gtttgtttat
      301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
      361 gtgtggggtc aaatagtttc atgttcccaa atggcccaaa actgacagtt taaacgctgt
      421 cttggaacct aatatgacaa aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt
      481 gaaatgctaa cggccagttg gtcaaaaaga aacttccaaa agtcgccata ccgtttgtct
      541 tgtttggtat tgattgacga atgctcaaaa ataatctcat taatgcttag cgcagtctct
      601 ctatcgcttc tgaacccggt ggcacctgtg ccgaaacgca aatggggaaa caacccgctt
      661 tttggatgat tatgcattgt cctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacaggcaa
      781 tatataaaca gaaggaagct gccctgtctt aaaccttttt ttttatcatc attattagct
      841 tactttcata attgcgactg gttccaattg acaagctttt gattttaacg acttttaacg
      901 acaacttgag aagatcaaaa aacaactaat tattcgaaac gatgttctct ccaattttgt
      961 ccttggaaat tattttagct ttggctactt tgcaatctgt cttcgctcga gaattccccg
     1021 ggatccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt
     1081 gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt
     1141 tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct
     1201 tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg
     1261 ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag
     1321 tacagaagat taagtgagaa gttcgtttgt gcaagcttat cgataagctt taatgcggta
     1381 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc
     1441 tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg
     1501 tactgccggg cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg
     1561 tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt
     1621 ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact
     1681 acgcgatcat ggcgaccaca cccgtcctgt ggatctatcg aatctaaatg taagttaaaa
     1741 tctctaaata attaaataag tcccagtttc tccatacgaa ccttaacagc attgcggtga
     1801 gcatctagac cttcaacagc agccagatcc atcactgctt ggccaatatg tttcagtccc
     1861 tcaggagtta cgtcttgtga agtgatgaac ttctggaagg ttgcagtgtt aactccgctg
     1921 tattgacggg catatccgta cgttggcaaa gtgtggttgg taccggagga gtaatctcca
     1981 caactctctg gagagtaggc accaacaaac acagatccag cgtgttgtac ttgatcaaca
     2041 taagaagaag cattctcgat ttgcaggatc aagtgttcag gagcgtactg attggacatt
     2101 tccaaagcct gctcgtaggt tgcaaccgat agggttgtag agtgtgcaat acacttgcgt
     2161 acaatttcaa cccttggcaa ctgcacagct tggttgtgaa cagcatcttc aattctggca
     2221 agctccttgt ctgtcatatc gacagccaac agaatcacct gggaatcaat accatgttca
     2281 gcttgagaca gaaggtctga ggcaacgaaa tctggatcag cgtatttatc agcaataact
     2341 agaacttcag aaggcccagc aggcatgtca atactacaca gggctgatgt gtcattttga
     2401 accatcatct tggcagcagt aacgaactgg tttcctggac caaatatttt gtcacactta
     2461 ggaacagttt ctgttccgta agccatagca gctactgcct gggcgcctcc tgctagcacg
     2521 atacacttag caccaacctt gtgggcaacg tagatgactt ctggggtaag ggtaccatcc
     2581 ttcttaggtg gagatgcaaa aacaatttct ttgcaaccag caactttggc aggaacaccc
     2641 agcatcaggg aagtggaagg cagaattgcg gttccaccag gaatatagag gccaactttc
     2701 tcaataggtc ttgcaaaacg agagcagact acaccagggc aagtctcaac ttgcaacgtc
     2761 tccgttagtt gagcttcatg gaatttcctg acgttatcta tagagagatc aatggctctc
     2821 ttaacgttat ctggcaattg cataagttcc tctgggaaag gagcttctaa cacaggtgtc
     2881 ttcaaagcga ctccatcaaa cttggcagtt agttctaaaa gggctttgtc accattttga
     2941 cgaacattgt cgacaattgg tttgactaat tccataatct gttccgtttt ctggatagga
     3001 cgacgaaggg catcttcaat ttcttgtgag gaggccttag aaacgtcaat tttgcacaat
     3061 tcaatacgac cttcagaagg gacttcttta ggtttggatt cttctttagg ttgttccttg
     3121 gtgtatcctg gcttggcatc tcctttcctt ctagtgacct ttagggactt catatccagg
     3181 tttctctcca cctcgtccaa cgtcacaccg tacttggcac atctaactaa tgcaaaataa
     3241 aataagtcag cacattccca ggctatatct tccttggatt tagcttctgc aagttcatca
     3301 gcttcctccc taattttagc gttcaacaaa acttcgtcgt caaataaccg tttggtataa
     3361 gaaccttctg gagcattgct cttacgatcc cacaaggtgg cttccatggc tctaagaccc
     3421 tttgattggc caaaacagga agtgcgttcc aagtgacaga aaccaacacc tgtttgttca
     3481 accacaaatt tcaagcagtc tccatcacaa tccaattcga tacccagcaa cttttgagtt
     3541 gctccagatg tagcaccttt ataccacaaa ccgtgacgac gagattggta gactccagtt
     3601 tgtgtcctta tagcctccgg aatagacttt ttggacgagt acaccaggcc caacgagtaa
     3661 ttagaagagt cagccaccaa agtagtgaat agaccatcgg ggcggtcagt agtcaaagac
     3721 gccaacaaaa tttcactgac agggaacttt ttgacatctt cagaaagttc gtattcagta
     3781 gtcaattgcc gagcatcaat aatggggatt ataccagaag caacagtgga agtcacatct
     3841 accaactttg cggtctcaga aaaagcataa acagttctac taccgccatt agtgaaactt
     3901 ttcaaatcgc ccagtggaga agaaaaaggc acagcgatac tagcattagc gggcaaggat
     3961 gcaactttat caaccagggt cctatagata accctagcgc ctgggatcat cctttggaca
     4021 actctttctg ccaaatctag gtccaaaatc acttcattga taccattatt gtacaacttg
     4081 agcaagttgt cgatcagctc ctcaaattgg tcctctgtaa cggatgactc aacttgcaca
     4141 ttaacttgaa gctcagtcga ttgagtgaac ttgatcaggt tgtgcagctg gtcagcagca
     4201 tagggaaaca cggcttttcc taccaaactc aaggaattat caaactctgc aacacttgcg
     4261 tatgcaggta gcaagggaaa tgtcatactt gaagtcggac agtgagtgta gtcttgagaa
     4321 attctgaagc cgtattttta ttatcagtga gtcagtcatc aggagatcct ctacgccgga
     4381 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
     4441 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
     4501 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
     4561 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
     4621 gagtcgcata agggagagcg tcgagtatct atgattggaa gtatgggaat ggtgataccc
     4681 gcattcttca gtgtcttgag gtctcctatc agattatgcc caactaaagc aaccggagga
     4741 ggagatttca tggtaaattt ctctgacttt tggtcatcag tagactcgaa ctgtgagact
     4801 atctcggtta tgacagcaga aatgtccttc ttggagacag taaatgaagt cccaccaata
     4861 aagaaatcct tgttatcagg aacaaacttc ttgtttcgaa ctttttcggt gccttgaact
     4921 ataaaatgta gagtggatat gtcgggtagg aatggagcgg gcaaatgctt accttctgga
     4981 ccttcaagag gtatgtaggg tttgtagata ctgatgccaa cttcagtgac aacgttgcta
     5041 tttcgttcaa accattccga atccagagaa atcaaagttg tttgtctact attgatccaa
     5101 gccagtgcgg tcttgaaact gacaatagtg tgctcgtgtt ttgaggtcat ctttgtatga
     5161 ataaatctag tctttgatct aaataatctt gacgagccaa ggcgataaat acccaaatct
     5221 aaaactcttt taaaacgtta aaaggacaag tatgtctgcc tgtattaaac cccaaatcag
     5281 ctcgtagtct gatcctcatc aacttgaggg gcactatctt gttttagaga aatttgcgga
     5341 gatgcgatat cgagaaaaag gtacgctgat tttaaacgtg aaatttatct caagatctct
     5401 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     5461 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     5521 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     5581 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     5641 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     5701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6061 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6301 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6541 aaggatcttc acctagatcc ttttacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     6601 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     6661 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     6721 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     6781 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     6841 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     6901 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6961 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     7681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     7741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7921 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7981 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     8041 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     8101 cgtatcacga ggccctttcg tcttcaagat ttctcatgtt tgacagctta tcatcgaatt
     8161 aattctcatg tttgacagct tatcatcgat aagctgactc atgttggtat tgtgaaatag
     8221 acgcagatcg ggaacactga aaaataacag ttattattcg
//
