LOCUS       40924_35374        5980 bp DNA     circular SYN 18-DEC-2018
DEFINITION  E.coli-K.pastoris shuttle vector pPpARG4, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5980)
  AUTHORS   Naatsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A.
  TITLE     Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform 
            Strain Generation for Gene Expression and Synthetic Biology
  JOURNAL   PLoS ONE 7 (6), E39720 (2012)
  PUBMED    22768112
REFERENCE   2  (bases 1 to 5980)
  AUTHORS   Naeaetsaari L, Mistlberger B, Ruth C, Hajek T, Hartner F, Glieder A.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-JAN-2012) Institute of Molecular Biotechnology, Graz 
            University of Technology, Petersgasse 14/2, Graz, Styria 8010, 
            Austria
REFERENCE   3  (bases 1 to 5980)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5980)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS ONE"; 
            date: "2012"; volume: "7"; issue: "6"; pages: "E39720"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (30-JAN-2012) Institute of Molecular Biotechnology, Graz University 
            of Technology, Petersgasse 14/2, Graz, Styria 8010, Austria"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: SeqMan v. 7.0.0
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        134..1068
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      1097..1343
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     regulatory      1369..2268
                     /note="ARG4 promoter; derived from Pichia pastoris"
                     /regulatory_class="promoter"
     CDS             2269..3666
                     /codon_start=1
                     /product="argininosuccinate lyase"
                     /label=argininosuccinate lyase
                     /note="ARG4; codon optimized"
                     /protein_id="AFJ20660.1"
                     /translation="MSNQEERLKLWGGRFTGATDPLMDLYNASLPYDKKMYKVDLEGTK
                     VYTEGLEKINLLTKDELSEIHRGLKLIEAEWAEGKFVEKPGDEDIHTANERRLGELIGR
                     GISGKVHTGRSRNDQVATDMRLYVRDNLTQLADYLKQFIQVIIKRAEQEIDVLMPGYTH
                     LQRAQPIRWSHWLSMYATYFTEDYERLNQIVKRLNKSPLGAGALAGHPYGIDREYIAER
                     LGFDSVIGNSLAAVSDRDFVVETMFWSSLFMNHISRFSEDLIIYSTGEFGFIKLADAYS
                     TGSSLMPQKKNPDSLELLRGKSGRCFGALAGFLMSIKSIPSTYNKDMQEDKEPLFDTLI
                     TVEHSILIASGVVSTLNIDAERMKNALTMDMLATDLADYLVRRGVPFRETHHISGECVR
                     QAEELNLSGIDQLSLEQLKSIDSRFEADVASTFDFEASVEKRTATGGTSKTAVLKQLDA
                     LNEKLES"
     regulatory      3668..4031
                     /note="ARG4 terminator; derived from Pichia pastoris"
                     /regulatory_class="terminator"
     regulatory      4032..4089
                     /label=EM72 synthetic promoter
                     /note="EM72 synthetic promoter"
                     /regulatory_class="promoter"
     CDS             4096..4953
                     /label=AmpR
                     /note="beta-lactamase"
     terminator      4963..5150
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     rep_origin      5314..5902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agatccaatt cccgctttga ctgcctgaaa tctccatcgc ctacaatgat gacatttgga
       61 tttggttgac tcatgttggt attgtgaaat agacgcagat cgggaacact gaaaaataca
      121 cagttattat tcagatctaa catccaaaga cgaaaggttg aatgaaacct ttttgccatc
      181 cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg ggatacacta
      241 gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac ccacttttgc
      301 catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc aattccttct
      361 attaggctac taacaccatg actttattag cctgtctatc ctggcccccc tggcgaggtt
      421 catgtttgtt tatttccgaa tgcaacaagc tccgcattac acccgaacat cactccagat
      481 gagggctttc tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc caaaactgac
      541 agtttaaacg ctgtcttgga acctaatatg acaaaagcgt gatctcatcc aagatgaact
      601 aagtttggtt cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc caaaagtcgg
      661 cataccgttt gtcttgtttg gtattgattg acgaatgctc aaaaataatc tcattaatgc
      721 ttagcgcagt ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa cgcaaatggg
      781 gaaacacccg ctttttggat gattatgcat tgtctccaca ttgtatgctt ccaagattct
      841 ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt ctaaccccta
      901 cttgacagca atatataaac agaaggaagc tgccctgtct taaccttttt ttttatcatc
      961 attattagct tactttcata attgcgactg gttccaattg acaagctttt gattttaacg
     1021 acttttaacg acaacttgag aagatcaaaa aacaactaat tattgaaaga attcactagt
     1081 ggcgcgccgc ggccgctcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt
     1141 catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt
     1201 ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agcagatgaa tatcttgtgg
     1261 taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc
     1321 agagtacaga agattaagtg agaccttcgt ttgtgcggat ccctcgagga ccgctttacc
     1381 tgctcttgga gacgtttact gtattcaaga atcattcaga gctgaaaaat ctcacaccag
     1441 aaggcattta tctgagtaca cccatattga ggccgaattg gccttcttga cttttgaaga
     1501 cctcttgtcc cacattgaga gagttatcac gaaaactgtc aaatatgtag ttaatgaccc
     1561 tgttgccggt cccttggtca aacaattgaa cccaggtttt gcagcaccag aactcccgtt
     1621 caagagaatg gagtacgtag aagcactgaa atggcttaat gatcacagta ttccaaacga
     1681 agaaggacaa cctttcaaat ttggtgacga cattgccgag gctgctgaga gaaagatgac
     1741 cgatactatt ggtgttccaa ttcttttgac tcgtttccca cttgaaatta agtcatttta
     1801 catgaaacgt tgcgccgacg atccaagagt tactgaatct gtagatgtgc tgatgccaac
     1861 tgttggagaa atcaccggtg gctctatgag gatcgactct ctcaatgaac tcctggaagg
     1921 ttttgaacgt gaaaagcttg acaaaagtgc gtactactgg ttcattgatc aacgaaaata
     1981 tggtacctgc cctcacggtg gttacggtct aggaacggaa cgtatcttag catggttgtg
     2041 cgacagattc actgtgaaag actgttcatt atacccacgt ttcactggga gatgtaagcc
     2101 ttaggtgttt taccctgatt agataataca ataaccaaca gaaatacgag aatctaaact
     2161 aatttcgatg attcattttt ctttttaccg cgctgcctct tttggcaatt ctttcaccta
     2221 tattctacct tctctttcct tttgttctaa acttattacc agctatctat gtctaaccag
     2281 gaagagagat tgaaactttg gggaggtaga ttcacaggag ctactgaccc tcttatggac
     2341 ctttacaatg catcacttcc ttatgacaag aagatgtaca aggttgatct tgaaggtaca
     2401 aaggtctaca ctgaaggact tgagaagatc aacttgctta ccaaagatga actttccgag
     2461 attcatcgag gattgaagct tatcgaagct gaatgggctg aaggcaagtt cgttgagaaa
     2521 cctggagatg aagacattca cactgccaat gaacgaagac ttggtgagtt gattggacgt
     2581 ggtatctctg gcaaagttca tacaggacgt tctagaaacg atcaggtcgc aacagacatg
     2641 cgattgtatg tcagagacaa tcttacgcaa cttgccgact acttgaaaca gttcattcaa
     2701 gtcatcatca agagagctga acaagagatt gatgtgctta tgcctggcta tacgcacctt
     2761 caacgagccc aacctattcg atggtcccat tggttgtcca tgtatgctac ttacttcacg
     2821 gaagactatg agcgtcttaa ccagattgtc aaacgactta acaagtcccc tcttggtgct
     2881 ggagcacttg ccggtcatcc ctatggcatt gatagagagt acattgccga aagacttggc
     2941 tttgattctg tgattggaaa ctctcttgct gcagttagcg atagagactt tgttgtggag
     3001 accatgtttt ggtcttcgct tttcatgaac cacatttcgc gattctctga ggatcttatc
     3061 atctacagca ctggtgagtt tggcttcatc aagcttgccg atgcttactc aaccggttct
     3121 tcccttatgc cacagaagaa gaatccagat tcgcttgaat tgcttcgagg taagtctgga
     3181 cgttgctttg gtgcacttgc tggcttcctt atgtctatca agtccattcc atctacctac
     3241 aacaaggaca tgcaagagga caaagagccc ttgtttgata ccttgattac tgttgagcat
     3301 tcgatcttga ttgcctctgg tgttgtgagc actcttaaca ttgatgctga acgaatgaag
     3361 aatgccctta caatggacat gcttgctact gatcttgccg actatcttgt cagacgaggt
     3421 gttccctttc gtgaaaccca tcacatttct ggtgaatgtg tgagacaagc cgaagagttg
     3481 aatctttccg gcattgatca actttccctt gagcagttga aaagcattga ttcacgtttc
     3541 gaagctgatg ttgcatcaac ctttgacttt gaggcttccg ttgagaaacg tacagcaact
     3601 ggtggaactt ccaagaccgc tgtgttgaaa cagcttgatg ccttgaatga gaaacttgaa
     3661 tcctaatagg ttttatactg agtttgttaa tgatacaata aactgttata gtacatacaa
     3721 ttgaaactct cttatctata ctgggggacc ttctcgcaga atggtataaa tatctactaa
     3781 ctgactgtcg tacggcctag gggtctcttc ttcgattatt tgcaggtcgg aacatccttc
     3841 gtctgatgcg gatctcctga gacaaagttc acgggtatct agtattctat cagcataaat
     3901 ggaggacctt tctaaactaa actttgaatc gtctccagca gcatcctcgc ataatccttt
     3961 tgtcatttcc tctatgtcta ttgtcactgt ggttggcgca tcaagagtcg tccttctgta
     4021 aaccggtaca ggtgttgaca ctttatactt ccggctcgta taatacgaca aggtgtaagg
     4081 aggactaaac ccgggatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4141 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4201 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4261 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4321 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4381 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4441 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4501 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4561 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4621 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4681 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4741 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4801 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4861 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4921 gctgagatag gtgcctcact gattaagcat tggtaaccta gggcgaattt cttatgattt
     4981 atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac
     5041 tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg
     5101 ctttctcagg tatagcatga ggtcgctctt attgaccaca cctctaccgg catgccgagc
     5161 aaatgcctgc aaatcgctcc ccatttcacc caattgtaga tatgctaact ccagcaatga
     5221 gttgatgaat ctcggtgtgt attttatgtc ctcagaggac aactgcaggt accactgagc
     5281 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5341 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5401 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5461 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5521 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5581 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5641 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5701 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5761 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5821 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5881 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5941 ttgctggcct tttgctcaca tgttctttcc tgcggtaccc
//
