LOCUS       Exported                8996 bp DNA     circular SYN 10-SEP-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8996)
  AUTHORS   11111111
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      1049..1295
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1708..4242)
                     /codon_start=1
                     /gene="Pichia pastoris HIS4"
                     /product="multifunctional enzyme, required for histidine 
                     biosynthesis"
                     /label=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(4655..5470)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     misc_feature    6741..6881
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7067..7655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7826..8686)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8687..8791)
                     /gene="bla"
                     /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 attcgaagga tcctacgtag aattccctag
      961 ggcggccgcg aattaattcg ccttagacat gactgttcct cagttcaagt tgggcactta
     1021 cgagaagacc ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag
     1081 agatgcaggc ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt
     1141 ttgtcatttt gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg
     1201 aatatcttgt ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc
     1261 ccactcctct tcagagtaca gaagattaag tgagacgttc gtttgtgcaa gcttatcgat
     1321 aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg
     1381 aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata
     1441 ggcttggtta tgccggtact gccgggcctc ttgcgggata tcgtccattc cgacagcatc
     1501 gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct atgcgcaccc
     1561 gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc ttcgctactt
     1621 ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtggat ctatcgaatc
     1681 taaatgtaag ttaaaatctc taaataatta aataagtccc agtttctcca tacgaacctt
     1741 aacagcattg cggtgagcat ctagaccttc aacagcagcc agatccatca ctgcttggcc
     1801 aatatgtttc agtccctcag gagttacgtc ttgtgaagtg atgaacttct ggaaggttgc
     1861 agtgttaact ccgctgtatt gacgggcata tccgtacgtt ggcaaagtgt ggttggtacc
     1921 ggaggagtaa tctccacaac tctctggaga gtaggcacca acaaacacag atccagcgtg
     1981 ttgtacttga tcaacataag aagaagcatt ctcgatttgc aggatcaagt gttcaggagc
     2041 gtactgattg gacatttcca aagcctgctc gtaggttgca accgataggg ttgtagagtg
     2101 tgcaatacac ttgcgtacaa tttcaaccct tggcaactgc acagcttggt tgtgaacagc
     2161 atcttcaatt ctggcaagct ccttgtctgt catatcgaca gccaacagaa tcacctggga
     2221 atcaatacca tgttcagctt gagacagaag gtctgaggca acgaaatctg gatcagcgta
     2281 tttatcagca ataactagaa cttcagaagg cccagcaggc atgtcaatac tacacagggc
     2341 tgatgtgtca ttttgaacca tcatcttggc agcagtaacg aactggtttc ctggaccaaa
     2401 tattttgtca cacttaggaa cagtttctgt tccgtaagcc atagcagcta ctgcctgggc
     2461 gcctcctgct agcacgatac acttagcacc aaccttgtgg gcaacgtaga tgacttctgg
     2521 ggtaagggta ccatccttct taggtggaga tgcaaaaaca atttctttgc aaccagcaac
     2581 tttggcagga acacccagca tcagggaagt ggaaggcaga attgcggttc caccaggaat
     2641 atagaggcca actttctcaa taggtcttgc aaaacgagag cagactacac cagggcaagt
     2701 ctcaacttgc aacgtctccg ttagttgagc ttcatggaat ttcctgacgt tatctataga
     2761 gagatcaatg gctctcttaa cgttatctgg caattgcata agttcctctg ggaaaggagc
     2821 ttctaacaca ggtgtcttca aagcgactcc atcaaacttg gcagttagtt ctaaaagggc
     2881 tttgtcacca ttttgacgaa cattgtcgac aattggtttg actaattcca taatctgttc
     2941 cgttttctgg ataggacgac gaagggcatc ttcaatttct tgtgaggagg ccttagaaac
     3001 gtcaattttg cacaattcaa tacgaccttc agaagggact tctttaggtt tggattcttc
     3061 tttaggttgt tccttggtgt atcctggctt ggcatctcct ttccttctag tgacctttag
     3121 ggacttcata tccaggtttc tctccacctc gtccaacgtc acaccgtact tggcacatct
     3181 aactaatgca aaataaaata agtcagcaca ttcccaggct atatcttcct tggatttagc
     3241 ttctgcaagt tcatcagctt cctccctaat tttagcgttc aacaaaactt cgtcgtcaaa
     3301 taaccgtttg gtataagaac cttctggagc attgctctta cgatcccaca aggtggcttc
     3361 catggctcta agaccctttg attggccaaa acaggaagtg cgttccaagt gacagaaacc
     3421 aacacctgtt tgttcaacca caaatttcaa gcagtctcca tcacaatcca attcgatacc
     3481 cagcaacttt tgagttgctc cagatgtagc acctttatac cacaaaccgt gacgacgaga
     3541 ttggtagact ccagtttgtg tccttatagc ctccggaata gactttttgg acgagtacac
     3601 caggcccaac gagtaattag aagagtcagc caccaaagta gtgaatagac catcggggcg
     3661 gtcagtagtc aaagacgcca acaaaatttc actgacaggg aactttttga catcttcaga
     3721 aagttcgtat tcagtagtca attgccgagc atcaataatg gggattatac cagaagcaac
     3781 agtggaagtc acatctacca actttgcggt ctcagaaaaa gcataaacag ttctactacc
     3841 gccattagtg aaacttttca aatcgcccag tggagaagaa aaaggcacag cgatactagc
     3901 attagcgggc aaggatgcaa ctttatcaac cagggtccta tagataaccc tagcgcctgg
     3961 gatcatcctt tggacaactc tttctgccaa atctaggtcc aaaatcactt cattgatacc
     4021 attattgtac aacttgagca agttgtcgat cagctcctca aattggtcct ctgtaacgga
     4081 tgactcaact tgcacattaa cttgaagctc agtcgattga gtgaacttga tcaggttgtg
     4141 cagctggtca gcagcatagg gaaacacggc ttttcctacc aaactcaagg aattatcaaa
     4201 ctctgcaaca cttgcgtatg caggtagcaa gggaaatgtc atacttgaag tcggacagtg
     4261 agtgtagtct tgagaaattc tgaagccgta tttttattat cagtgagtca gtcatcagga
     4321 gatcctctac gccggacgca tcgtggccga cctgcagggg ggggggggcg ctgaggtctg
     4381 cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag
     4441 aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg
     4501 aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc
     4561 aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc
     4621 tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat
     4681 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4741 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4801 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4861 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     4921 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4981 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     5041 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     5101 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     5161 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     5221 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     5281 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     5341 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     5401 ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa
     5461 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     5521 atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt
     5581 cccccccccc cctgcaggtc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     5641 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     5701 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     5761 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     5821 taatgcagga gtcgcataag ggagagcgtc gagtatctat gattggaagt atgggaatgg
     5881 tgatacccgc attcttcagt gtcttgaggt ctcctatcag attatgccca actaaagcaa
     5941 ccggaggagg agatttcatg gtaaatttct ctgacttttg gtcatcagta gactcgaact
     6001 gtgagactat ctcggttatg acagcagaaa tgtccttctt ggagacagta aatgaagtcc
     6061 caccaataaa gaaatccttg ttatcaggaa caaacttctt gtttcgaact ttttcggtgc
     6121 cttgaactat aaaatgtaga gtggatatgt cgggtaggaa tggagcgggc aaatgcttac
     6181 cttctggacc ttcaagaggt atgtagggtt tgtagatact gatgccaact tcagtgacaa
     6241 cgttgctatt tcgttcaaac cattccgaat ccagagaaat caaagttgtt tgtctactat
     6301 tgatccaagc cagtgcggtc ttgaaactga caatagtgtg ctcgtgtttt gaggtcatct
     6361 ttgtatgaat aaatctagtc tttgatctaa ataatcttga cgagccagac gataatacca
     6421 atctaaactc tttaaacgtt aaaggacaag tatgtctgcc tgtattaaac cccaaatcag
     6481 ctcgtagtct gatcctcatc aacttgaggg gcactatctt gttttagaga aatttgcgga
     6541 gatgcgatat cgagaaaaag gtacgctgat tttaaacgtg aaatttatct caagatctgc
     6601 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     6661 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     6721 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     6781 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     6841 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     6901 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6961 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     7021 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     7081 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     7141 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     7201 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     7261 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     7321 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     7381 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     7441 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     7501 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7561 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7621 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7681 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7741 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7801 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7861 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7921 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7981 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     8041 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     8101 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     8161 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     8221 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     8281 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     8341 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     8401 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     8461 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     8521 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     8581 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     8641 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     8701 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8761 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     8821 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     8881 ttcgtcttca agaattaatt ctcatgtttg acagcttatc atcgataagc tgactcatgt
     8941 tggtattgtg aaatagacgc agatcgggaa cactgaaaaa taacagttat tattcg
//
