LOCUS       Exported                9911 bp DNA     circular SYN 08-DEC-2025
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9911)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9911)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             944..1660
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      1751..1997
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(2410..4944)
                     /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(5361..6380)
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     misc_feature    6765..7513
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7656..7796
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7982..8570)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8744..9601)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9602..9706)
                     /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 tccatggtga gcaagggcga
      961 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     1021 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     1081 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1141 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1201 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1261 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1321 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1381 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1441 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1501 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1561 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1621 cgccgccggg atcactctcg gcatggacga gctgtacaag taagcggccg cgaattaatt
     1681 cgccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc
     1741 tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt
     1801 tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc
     1861 tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg
     1921 tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta
     1981 cagaagatta agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt
     2041 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
     2101 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
     2161 ctgccgggcc tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg
     2221 ctgctagcgc tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc
     2281 gaccgctttg gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac
     2341 gcgatcatgg cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc
     2401 tctaaataat taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc
     2461 atctagacct tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc
     2521 aggagttacg tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta
     2581 ttgacgggca tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca
     2641 actctctgga gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata
     2701 agaagaagca ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc
     2761 caaagcctgc tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac
     2821 aatttcaacc cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag
     2881 ctccttgtct gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc
     2941 ttgagacaga aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag
     3001 aacttcagaa ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac
     3061 catcatcttg gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg
     3121 aacagtttct gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat
     3181 acacttagca ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt
     3241 cttaggtgga gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag
     3301 catcagggaa gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc
     3361 aataggtctt gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc
     3421 cgttagttga gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt
     3481 aacgttatct ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt
     3541 caaagcgact ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg
     3601 aacattgtcg acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg
     3661 acgaagggca tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc
     3721 aatacgacct tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt
     3781 gtatcctggc ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt
     3841 tctctccacc tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa
     3901 taagtcagca cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc
     3961 ttcctcccta attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga
     4021 accttctgga gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt
     4081 tgattggcca aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac
     4141 cacaaatttc aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc
     4201 tccagatgta gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg
     4261 tgtccttata gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt
     4321 agaagagtca gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc
     4381 caacaaaatt tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt
     4441 caattgccga gcatcaataa tggggattat accagaagca acagtggaag tcacatctac
     4501 caactttgcg gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt
     4561 caaatcgccc agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc
     4621 aactttatca accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac
     4681 tctttctgcc aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag
     4741 caagttgtcg atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt
     4801 aacttgaagc tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata
     4861 gggaaacacg gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta
     4921 tgcaggtagc aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat
     4981 tctgaagccg tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg
     5041 catcgtggcc gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt
     5101 tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg
     5161 gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac
     5221 ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg
     5281 atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac
     5341 caattaacca attctgatta ttcctttgcc ctcggacgag tgctggggcg tcggtttcca
     5401 ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt
     5461 tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc
     5521 caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg
     5581 cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct ccgctcgaag
     5641 tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc
     5701 tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca
     5761 ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg
     5821 cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg
     5881 tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca
     5941 cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg
     6001 ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag aacagcgggc
     6061 agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag
     6121 gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag cgcggccgat
     6181 gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc
     6241 aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag
     6301 agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc
     6361 gcggtgagtt caggcttttt acccataaca ccccttgtat tactgtttat gtaagcagac
     6421 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     6481 gacacaacgt ggctttcccc cccccccctg caggtcggca tcaccggcgc cacaggtgcg
     6541 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     6601 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     6661 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     6721 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgagt atctatgatt
     6781 ggaagtatgg gaatggtgat acccgcattc ttcagtgtct tgaggtctcc tatcagatta
     6841 tgcccaacta aagcaaccgg aggaggagat ttcatggtaa atttctctga cttttggtca
     6901 tcagtagact cgaactgtga gactatctcg gttatgacag cagaaatgtc cttcttggag
     6961 acagtaaatg aagtcccacc aataaagaaa tccttgttat caggaacaaa cttcttgttt
     7021 cgaacttttt cggtgccttg aactataaaa tgtagagtgg atatgtcggg taggaatgga
     7081 gcgggcaaat gcttaccttc tggaccttca agaggtatgt agggtttgta gatactgatg
     7141 ccaacttcag tgacaacgtt gctatttcgt tcaaaccatt ccgaatccag agaaatcaaa
     7201 gttgtttgtc tactattgat ccaagccagt gcggtcttga aactgacaat agtgtgctcg
     7261 tgttttgagg tcatctttgt atgaataaat ctagtctttg atctaaataa tcttgacgag
     7321 ccagacgata ataccaatct aaactcttta aacgttaaag gacaagtatg tctgcctgta
     7381 ttaaacccca aatcagctcg tagtctgatc ctcatcaact tgaggggcac tatcttgttt
     7441 tagagaaatt tgcggagatg cgatatcgag aaaaaggtac gctgatttta aacgtgaaat
     7501 ttatctcaag atctctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     7561 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     7621 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     7681 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     7741 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     7801 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     7861 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     7921 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     7981 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     8041 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     8101 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     8161 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     8221 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     8281 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     8341 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     8401 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     8461 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     8521 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     8581 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     8641 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     8701 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     8761 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     8821 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     8881 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     8941 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     9001 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     9061 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     9121 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     9181 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     9241 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     9301 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     9361 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     9421 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     9481 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     9541 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     9601 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     9661 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     9721 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     9781 gtatcacgag gccctttcgt cttcaagaat taattctcat gtttgacagc ttatcatcga
     9841 taagctgact catgttggta ttgtgaaata gacgcagatc gggaacactg aaaaataaca
     9901 gttattattc g
//
