LOCUS       Exported                8016 bp DNA     circular SYN 27-FEB-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8016)
  AUTHORS   11111111
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             949..1215
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     terminator      1321..1567
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1980..4514)
                     /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"
     misc_feature    5761..5901
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(6087..6675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6846..7706)
                     /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(7707..7811)
                     /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 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 cgaattaatt cgccttagac
     1261 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa
     1321 tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt
     1381 ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc
     1441 ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa
     1501 tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta
     1561 agtgagacgt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt
     1621 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc
     1681 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc
     1741 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc
     1801 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg
     1861 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg
     1921 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat
     1981 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct
     2041 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg
     2101 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca
     2161 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga
     2221 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca
     2281 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc
     2341 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc
     2401 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct
     2461 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga
     2521 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa
     2581 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg
     2641 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct
     2701 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca
     2761 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga
     2821 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa
     2881 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt
     2941 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga
     3001 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct
     3061 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact
     3121 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg
     3181 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca
     3241 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct
     3301 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc
     3361 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc
     3421 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca
     3481 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta
     3541 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga
     3601 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca
     3661 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc
     3721 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta
     3781 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata
     3841 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca
     3901 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt
     3961 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga
     4021 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg
     4081 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc
     4141 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca
     4201 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc
     4261 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg
     4321 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc
     4381 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg
     4441 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc
     4501 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg
     4561 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc
     4621 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     4681 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     4741 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     4801 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     4861 ggagagcgtc gagtatctat gattggaagt atgggaatgg tgatacccgc attcttcagt
     4921 gtcttgaggt ctcctatcag attatgccca actaaagcaa ccggaggagg agatttcatg
     4981 gtaaatttct ctgacttttg gtcatcagta gactcgaact gtgagactat ctcggttatg
     5041 acagcagaaa tgtccttctt ggagacagta aatgaagtcc caccaataaa gaaatccttg
     5101 ttatcaggaa caaacttctt gtttcgaact ttttcggtgc cttgaactat aaaatgtaga
     5161 gtggatatgt cgggtaggaa tggagcgggc aaatgcttac cttctggacc ttcaagaggt
     5221 atgtagggtt tgtagatact gatgccaact tcagtgacaa cgttgctatt tcgttcaaac
     5281 cattccgaat ccagagaaat caaagttgtt tgtctactat tgatccaagc cagtgcggtc
     5341 ttgaaactga caatagtgtg ctcgtgtttt gaggtcatct ttgtatgaat aaatctagtc
     5401 tttgatctaa ataatcttga cgagccagac gataatacca atctaaactc tttaaacgtt
     5461 aaaggacaag tatgtctgcc tgtattaaac cccaaatcag ctcgtagtct gatcctcatc
     5521 aacttgaggg gcactatctt gttttagaga aatttgcgga gatgcgatat cgagaaaaag
     5581 gtacgctgat tttaaacgtg aaatttatct caagatctgc tgcctcgcgc gtttcggtga
     5641 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     5701 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     5761 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     5821 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     5881 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5941 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6001 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6061 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     6541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6661 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6721 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6781 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6841 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6901 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6961 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7021 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7081 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7141 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7201 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7261 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7321 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7381 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7441 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     7501 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7561 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7621 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7681 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7741 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7801 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7861 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt
     7921 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc
     7981 agatcgggaa cactgaaaaa taacagttat tattcg
//
