LOCUS       40924_34550        9294 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9294)
  AUTHORS   caoheibi
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9294)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9294
                     /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"
     CDS             1246..1263
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1339..1585
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1998..4532)
                     /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(4949..5761)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6140..6896
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7039..7179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7365..7953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8127..8984)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8985..9089)
                     /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 aaagtcgcca 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 cgaatcatca tcaccatcac
     1261 cattaattcg ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc
     1321 ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc
     1381 ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt
     1441 gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt
     1501 ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct
     1561 tcagagtaca gaagattaag tgagaagttc gtttgtgcaa gcttatcgat aagctttaat
     1621 gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg aaatctaaca
     1681 atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata ggcttggtta
     1741 tgccggtact gccgggcctc ttgcgggata tcgtccattc cgacagcatc gccagtcact
     1801 atggcgtgct gctagcgcta tatgcgttga tgcaatttct atgcgcaccc gttctcggag
     1861 cactgtccga ccgctttggc cgccgcccag tcctgctcgc ttcgctactt ggagccacta
     1921 tcgactacgc gatcatggcg accacacccg tcctgtggat ctatcgaatc taaatgtaag
     1981 ttaaaatctc taaataatta aataagtccc agtttctcca tacgaacctt aacagcattg
     2041 cggtgagcat ctagaccttc aacagcagcc agatccatca ctgcttggcc aatatgtttc
     2101 agtccctcag gagttacgtc ttgtgaagtg atgaacttct ggaaggttgc agtgttaact
     2161 ccgctgtatt gacgggcata tccgtacgtt ggcaaagtgt ggttggtacc ggaggagtaa
     2221 tctccacaac tctctggaga gtaggcacca acaaacacag atccagcgtg ttgtacttga
     2281 tcaacataag aagaagcatt ctcgatttgc aggatcaagt gttcaggagc gtactgattg
     2341 gacatttcca aagcctgctc gtaggttgca accgataggg ttgtagagtg tgcaatacac
     2401 ttgcgtacaa tttcaaccct tggcaactgc acagcttggt tgtgaacagc atcttcaatt
     2461 ctggcaagct ccttgtctgt catatcgaca gccaacagaa tcacctggga atcaatacca
     2521 tgttcagctt gagacagaag gtctgaggca acgaaatctg gatcagcgta tttatcagca
     2581 ataactagaa cttcagaagg cccagcaggc atgtcaatac tacacagggc tgatgtgtca
     2641 ttttgaacca tcatcttggc agcagtaacg aactggtttc ctggaccaaa tattttgtca
     2701 cacttaggaa cagtttctgt tccgtaagcc atagcagcta ctgcctgggc gcctcctgct
     2761 agcacgatac acttagcacc aaccttgtgg gcaacgtaga tgacttctgg ggtaagggta
     2821 ccatccttct taggtggaga tgcaaaaaca atttctttgc aaccagcaac tttggcagga
     2881 acacccagca tcagggaagt ggaaggcaga attgcggttc caccaggaat atagaggcca
     2941 actttctcaa taggtcttgc aaaacgagag cagactacac cagggcaagt ctcaacttgc
     3001 aacgtctccg ttagttgagc ttcatggaat ttcctgacgt tatctataga gagatcaatg
     3061 gctctcttaa cgttatctgg caattgcata agttcctctg ggaaaggagc ttctaacaca
     3121 ggtgtcttca aagcgactcc atcaaacttg gcagttagtt ctaaaagggc tttgtcacca
     3181 ttttgacgaa cattgtcgac aattggtttg actaattcca taatctgttc cgttttctgg
     3241 ataggacgac gaagggcatc ttcaatttct tgtgaggagg ccttagaaac gtcaattttg
     3301 cacaattcaa tacgaccttc agaagggact tctttaggtt tggattcttc tttaggttgt
     3361 tccttggtgt atcctggctt ggcatctcct ttccttctag tgacctttag ggacttcata
     3421 tccaggtttc tctccacctc gtccaacgtc acaccgtact tggcacatct aactaatgca
     3481 aaataaaata agtcagcaca ttcccaggct atatcttcct tggatttagc ttctgcaagt
     3541 tcatcagctt cctccctaat tttagcgttc aacaaaactt cgtcgtcaaa taaccgtttg
     3601 gtataagaac cttctggagc attgctctta cgatcccaca aggtggcttc catggctcta
     3661 agaccctttg attggccaaa acaggaagtg cgttccaagt gacagaaacc aacacctgtt
     3721 tgttcaacca caaatttcaa gcagtctcca tcacaatcca attcgatacc cagcaacttt
     3781 tgagttgctc cagatgtagc acctttatac cacaaaccgt gacgacgaga ttggtagact
     3841 ccagtttgtg tccttatagc ctccggaata gactttttgg acgagtacac caggcccaac
     3901 gagtaattag aagagtcagc caccaaagta gtgaatagac catcggggcg gtcagtagtc
     3961 aaagacgcca acaaaatttc actgacaggg aactttttga catcttcaga aagttcgtat
     4021 tcagtagtca attgccgagc atcaataatg gggattatac cagaagcaac agtggaagtc
     4081 acatctacca actttgcggt ctcagaaaaa gcataaacag ttctactacc gccattagtg
     4141 aaacttttca aatcgcccag tggagaagaa aaaggcacag cgatactagc attagcgggc
     4201 aaggatgcaa ctttatcaac cagggtccta tagataaccc tagcgcctgg gatcatcctt
     4261 tggacaactc tttctgccaa atctaggtcc aaaatcactt cattgatacc attattgtac
     4321 aacttgagca agttgtcgat cagctcctca aattggtcct ctgtaacgga tgactcaact
     4381 tgcacattaa cttgaagctc agtcgattga gtgaacttga tcaggttgtg cagctggtca
     4441 gcagcatagg gaaacacggc ttttcctacc aaactcaagg aattatcaaa ctctgcaaca
     4501 cttgcgtatg caggtagcaa gggaaatgtc atacttgaag tcggacagtg agtgtagtct
     4561 tgagaaattc tgaagccgta tttttattat cagtgagtca gtcatcagga gatcctctac
     4621 gccggacgca tcgtggccga cctgcagggg gggggggggc gctgaggtct gcctcgtgaa
     4681 gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg
     4741 gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc
     4801 tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca
     4861 aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt
     4921 gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca
     4981 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     5041 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     5101 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     5161 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt
     5221 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     5281 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     5341 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     5401 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     5461 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     5521 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     5581 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     5641 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     5701 catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca
     5761 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat
     5821 ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tccccccccc
     5881 ccctgcaggt cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     5941 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     6001 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     6061 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     6121 agtcgcataa gggagagcgt cgagtatcta tgattggaag tatgggaatg gtgatacccg
     6181 cattcttcag tgtcttgagg tctcctatca gattatgccc aactaaagca accggaggag
     6241 gagatttcat ggtaaatttc tctgactttt ggtcatcagt agactcgaac tgtgagacta
     6301 tctcggttat gacagcagaa atgtccttct tggagacagt aaatgaagtc ccaccaataa
     6361 agaaatcctt gttatcagga acaaacttct tgtttcgaac tttttcggtg ccttgaacta
     6421 taaaatgtag agtggatatg tcgggtagga atggagcggg caaatgctta ccttctggac
     6481 cttcaagagg tatgtagggt ttgtagatac tgatgccaac ttcagtgaca acgttgctat
     6541 ttcgttcaaa ccattccgaa tccagagaaa tcaaagttgt ttgtctacta ttgatccaag
     6601 ccagtgcggt cttgaaactg acaatagtgt gctcgtgttt tgaggtcatc tttgtatgaa
     6661 taaatctagt ctttgatcta aataatcttg acgagccaag gcgataaata cccaaatcta
     6721 aaactctttt aaaacgttaa aaggacaagt atgtctgcct gtattaaacc ccaaatcagc
     6781 tcgtagtctg atcctcatca acttgagggg cactatcttg ttttagagaa atttgcggag
     6841 atgcgatatc gagaaaaagg tacgctgatt ttaaacgtga aatttatctc aagatctctg
     6901 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6961 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7021 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     7081 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     7141 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     7201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     7261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     7321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     7381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     7441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa
     7561 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7801 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     8041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     8101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     8161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     8221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     8281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     8341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     8401 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     8461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     8761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     9001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     9061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     9121 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     9181 cgtcttcaag aattaattct catgtttgac agcttatcat cgataagctg actcatgttg
     9241 gtattgtgaa atagacgcag atcgggaaca ctgaaaaata acagttatta ttcg
//
