LOCUS       sequence-232357-        4040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  low copy plasmid, neg. control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4040)
  AUTHORS   Steiger MG, Rassinger A, Mattanovich D, Sauer M
  TITLE     Engineering of the citrate exporter protein enables high citric acid
            production in Aspergillus niger.
  JOURNAL   Metab Eng. 2019 Mar;52:224-231. doi: 10.1016/j.ymben.2018.12.004. 
            Epub 2018 Dec 13.
  PUBMED    30553933
REFERENCE   2  (bases 1 to 4040)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Metab 
            Eng."; date: "2019-03"; volume: "52:224-231. doi"; pages: " 
            10.1016/j.ymben.2018.12.004. Epub 2018 Dec 13"
COMMENT     Sequence Label: BB3_arscen_URA3_Hygro
FEATURES             Location/Qualifiers
     source          1..4040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        748..963
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             964..1767
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     gene            1949..3524
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1949..2292
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2293..3321
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2293..3321
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      3327..3524
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_feature    3536..4039
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 cgctgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtgg agcgcaaaaa accccgcccc tgacagggcg gggttttttc
      721 gcgatcggag cagggtccat aaagcttttc aattcatctt ttttttttta ttcttttttt
      781 tgattccggt ttccttgaaa tttttttgat tcggtaatct ccgagcagaa ggaagaacga
      841 aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga
      901 aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa
      961 atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc
     1021 aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt
     1081 accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa
     1141 acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca
     1201 ttatccgcca agtacaattt tttactcttc gaggacagaa aatttgctga cattggtaat
     1261 acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg
     1321 aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcggaagaa
     1381 gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta
     1441 gctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt
     1501 atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt
     1561 atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca gtatagaacc
     1621 gtggatgatg tggtgtctac aggatctgac attattattg ttggaagagg actatttgca
     1681 aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat
     1741 ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta
     1801 aactcacaaa ttagagcttc aatttaatta tatcagttat tacccgggaa tctcggcgct
     1861 ccggggaggc gtcttcggat ccctcgagga agacgccgct aattggaggc gtcttcggat
     1921 ccctcgagga agacgccgct agctcatgga catggaggcc cagaataccc tccttgacag
     1981 tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca
     2041 tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa
     2101 ttacggctcc tcgctgcaga cgtgcgagca gggaaacgct cccctcacag acgcgttgaa
     2161 ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt
     2221 cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga
     2281 acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     2341 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     2401 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     2461 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     2521 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     2581 cagggtgtca cgttgcaaga cctccctgaa accgaactgc ccgctgttct gcagccggtc
     2641 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     2701 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     2761 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     2821 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     2881 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     2941 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3001 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3061 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3121 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     3181 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     3241 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     3301 cgtccgaggg caaaggaata atcagtactg acaataaaaa gattcttgtt ttcaagaact
     3361 tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga
     3421 tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag
     3481 taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actggcttcc ttttcatcac
     3541 gtgctataaa aataattata atttaaattt tttaatataa atatataaat taaaaataga
     3601 aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct
     3661 agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca ggtattaatg
     3721 ccgaattgtt tcatcttgtc tgtgtagaac accacacacg aaaatcctgt gattttacat
     3781 tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat
     3841 atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca
     3901 ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa aacataaaaa
     3961 taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt
     4021 aagttacagg caagcgatcg
//
