LOCUS       pHIL-D2.        8210 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Pichia pastoris HIS4 vector for methanol-inducible intracellular 
            expression of a protein.
ACCESSION   .
VERSION     .
KEYWORDS    pHIL-D2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8210)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8210)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     The gene of interest can be inserted at the unique EcoRI site.
FEATURES             Location/Qualifiers
     source          1..8210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..953
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      1031..1277
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1690..4224)
                     /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    4579..5335
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     promoter        5582..5686
                     /label=AmpR promoter
     CDS             5687..6544
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      complement(6589..7044)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     rep_origin      7155..7743
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(7929..8069)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 agatcgcggc cgcgatctaa catccaaaga cgaaaggttg aatgaaacct ttttgccatc
       61 cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg ggatacacta
      121 gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac ccacttttgc
      181 catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc aattccttct
      241 attaggctac taacaccatg actttattag cctgtctatc ctggcccccc tggcgaggtc
      301 atgtttgttt atttccgaat gcaacaagct ccgcattaca cccgaacatc actccagatg
      361 agggctttct gagtgtgggg tcaaatagtt tcatgttccc aaatggccca aaactgacag
      421 tttaaacgct gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa
      481 gtttggttcg ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca
      541 taccgtttgt cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt
      601 agcgcagtct ctctatcgct tctgaacccg gtggcacctg tgccgaaacg caaatgggga
      661 aacaacccgc tttttggatg attatgcatt gtcctccaca ttgtatgctt ccaagattct
      721 ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt ctaaccccta
      781 cttgacaggc aatatataaa cagaaggaag ctgccctgtc ttaaaccttt ttttttatca
      841 tcattattag cttactttca taattgcgac tggttccaat tgacaagctt ttgattttaa
      901 cgacttttaa cgacaacttg agaagatcaa aaaacaacta attattcgaa acgaggaatt
      961 cgccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc
     1021 tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt
     1081 tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc
     1141 tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg
     1201 tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta
     1261 cagaagatta agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt
     1321 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
     1381 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
     1441 ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg
     1501 ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc
     1561 gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac
     1621 gcgatcatgg cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc
     1681 tctaaataat taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc
     1741 atctagacct tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc
     1801 aggagttacg tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta
     1861 ttgacgggca tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca
     1921 actctctgga gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata
     1981 agaagaagca ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc
     2041 caaagcctgc tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac
     2101 aatttcaacc cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag
     2161 ctccttgtct gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc
     2221 ttgagacaga aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag
     2281 aacttcagaa ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac
     2341 catcatcttg gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg
     2401 aacagtttct gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat
     2461 acacttagca ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt
     2521 cttaggtgga gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag
     2581 catcagggaa gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc
     2641 aataggtctt gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc
     2701 cgttagttga gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt
     2761 aacgttatct ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt
     2821 caaagcgact ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg
     2881 aacattgtcg acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg
     2941 acgaagggca tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc
     3001 aatacgacct tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt
     3061 gtatcctggc ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt
     3121 tctctccacc tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa
     3181 taagtcagca cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc
     3241 ttcctcccta attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga
     3301 accttctgga gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt
     3361 tgattggcca aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac
     3421 cacaaatttc aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc
     3481 tccagatgta gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg
     3541 tgtccttata gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt
     3601 agaagagtca gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc
     3661 caacaaaatt tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt
     3721 caattgccga gcatcaataa tggggattat accagaagca acagtggaag tcacatctac
     3781 caactttgcg gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt
     3841 caaatcgccc agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc
     3901 aactttatca accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac
     3961 tctttctgcc aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag
     4021 caagttgtcg atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt
     4081 aacttgaagc tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata
     4141 gggaaacacg gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta
     4201 tgcaggtagc aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat
     4261 tctgaagccg tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg
     4321 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     4381 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     4441 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     4501 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     4561 gtcgcataag ggagagcgtc gagtatctat gattggaagt atgggaatgg tgatacccgc
     4621 attcttcagt gtcttgaggt ctcctatcag attatgccca actaaagcaa ccggaggagg
     4681 agatttcatg gtaaatttct ctgacttttg gtcatcagta gactcgaact gtgagactat
     4741 ctcggttatg acagcagaaa tgtccttctt ggagacagta aatgaagtcc caccaataaa
     4801 gaaatccttg ttatcaggaa caaacttctt gtttcgaact ttttcggtgc cttgaactat
     4861 aaaatgtaga gtggatatgt cgggtaggaa tggagcgggc aaatgcttac cttctggacc
     4921 ttcaagaggt atgtagggtt tgtagatact gatgccaact tcagtgacaa cgttgctatt
     4981 tcgttcaaac cattccgaat ccagagaaat caaagttgtt tgtctactat tgatccaagc
     5041 cagtgcggtc ttgaaactga caatagtgtg ctcgtgtttt gaggtcatct ttgtatgaat
     5101 aaatctagtc tttgatctaa ataatcttga cgagccaagg cgataaatac ccaaatctaa
     5161 aactctttta aaacgttaaa aggacaagta tgtctgcctg tattaaaccc caaatcagct
     5221 cgtagtctga tcctcatcaa cttgaggggc actatcttgt tttagagaaa tttgcggaga
     5281 tgcgatatcg agaaaaaggt acgctgattt taaacgtgaa atttatctca agatcgcggc
     5341 cgcgatctcg aataataact gttatttttc agtgttcccg atctgcgtct atttcacaat
     5401 accaacatga gtcagcttat cgatgataag ctgtcaaaca tgagaattaa ttcgatgata
     5461 agctgtcaaa catgagaaat cttgaagacg aaagggcctc gtgatacgcc tatttttata
     5521 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5641 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5701 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5761 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5821 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5881 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     5941 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6001 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6061 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6121 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6181 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     6241 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6301 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6361 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6421 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6481 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6541 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa attgtaaacg
     6601 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6661 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6721 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6781 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6841 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6901 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6961 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     7021 ttaatgcgcc gctacagggc gcgtaaaagg atctaggtga agatcctttt tgataatctc
     7081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7261 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     7321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagcattg agaaagcgcc
     7561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7921 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     7981 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     8041 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     8101 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     8161 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
//
