LOCUS       sequence-69091-e        7535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate-inducible expression of 3xFLAG-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7535)
  AUTHORS   Kaczmarczyk A, Vorholt JA, Francez-Charlot A
  TITLE     Cumate-inducible gene expression system for sphingomonads and other 
            Alphaproteobacteria.
  JOURNAL   Appl Environ Microbiol. 2013 Nov;79(21):6795-802. doi: 
            10.1128/AEM.02296-13. Epub 2013 Aug 30.
  PUBMED    23995928
REFERENCE   2  (bases 1 to 7535)
  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"; doi: 
            "10.1128/AEM.02296-13"; journalName: "Appl Environ Microbiol"; date:
            "2013-11"; volume: "79"; issue: "21"; pages: "6795-802"
COMMENT     Sequence Label: pQF
FEATURES             Location/Qualifiers
     source          1..7535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1594..1613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1755..2366)
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     protein_bind    2443..2470
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     protein_bind    2513..2540
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     CDS             2565..2630
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2685..2750
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2822..2839)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2822..2838)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2831..2853)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3047..3066)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3166..3188
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3226..3244)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4030..5229
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     CDS             complement(5460..6608)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(6880..7251)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7284..7393
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg
      721 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      781 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      841 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
      901 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
      961 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1021 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1081 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1141 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1201 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1261 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1321 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1381 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1441 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1501 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1561 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1621 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1681 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1741 ttttgctcac atgttcagcg cttgaacttg gcgtagcgct cgcgggcgat ttccagggtc
     1801 gagttgcgga cgcgctcgaa gcgctccttg tccttctgcc acagcgagcg gaccgccagg
     1861 ccgcggaccg agttgaagat cagccacagg atgtcctccg cgtcgtcgcg gctcaggccg
     1921 cgcgacacca gcacgcccag ccacatatcc tcgacgacga agcggttgcg ctcgacggtg
     1981 cgctggatgc cctcgcgcag cgccgggtcg cggtcggcgg ccacgatcag gtccagcgag
     2041 atcgagaagt cgtcgtccag gaagaactcg gcggcgtcgt ccagcatctg ctggatgacg
     2101 tcgtcctcgg gcttcagctt ggccaggcgg gcgcggctgc gctcggtgat ctgctcgtac
     2161 agccactcga aggtggccag cagcagttcc agcttggtcg ggaagtggtg cgactgggcg
     2221 ccgcggctga cgccggcggc gcccgggacg tcggcgatgc ggaagccggc atagcccttc
     2281 tcgcgcagga cgcccagggc ggcggcgatc agcttgccct gggtttccat cgcgcgctcg
     2341 gcctgggtgc ggcgcttcgg cgacatgatg accattcggg gtctctccct gctagcatca
     2401 gggttattgt atcatgagcg gatacatatg tcaatgtacc ggaacaaaca gacaatctgg
     2461 tctgtttgta catggttgac ggatcgccgc gaagccgcta actgcgccgg ttaacaaaca
     2521 gacaatctgg tctgtttgta actagtagag gaagcttccg catggactac aaggaccacg
     2581 acggcgacta taaggaccac gacatcgact acaaggatga cgacgacaag cttattaata
     2641 tgactctaga agatctcgag gtaccgagct ccaattggga tccggactac aaggaccacg
     2701 acggcgacta taaggaccac gacatcgact acaaggatga cgacgacaag tgaggatccg
     2761 gtgaagtgac ccgcaaaaaa aaaggccagg ggatcgctcc cccggccttt tttttgaatt
     2821 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2881 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2941 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     3001 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     3061 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3121 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3181 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3241 tatttttata ggttaatgtc atgataataa tggtttctta gcaccctttc tcggtccttc
     3301 aacgttcctg acaacgagcc tccttttcgc caatccatcg acaatcaccg cgagtccctg
     3361 ctcgaacgct gcgtccggac cggcttcgtc gaaggcgtct atcgcggccc gcaacagcgg
     3421 cgagagcgga gcctgttcaa cggtgccgcc gcgctcgccg gcatcgctgt cgccggcctg
     3481 ctcctcaagc acggccccaa cagtgaagta gctgattgtc atcagcgcat tgacggcgtc
     3541 cccggccgaa aaacccgcct cgcagaggaa gcgaagctgc gcgtcggccg tttccatctg
     3601 cggtgcgccc ggtcgcgtgc cggcatggat gcgcgcgcca tcgcggtagg cgagcagcgc
     3661 ctgcctgaag ctgcgggcat tcccgatcag aaatgagcgc cagtcgtcgt cggctctcgg
     3721 caccgaatgc gtatgattct ccgccagcat ggcttcggcc agtgcgtcga gcagcgcccg
     3781 cttgttcctg aagtgccagt aaagcgccgg ctgctgaacc cccaaccgtt ccgccagttt
     3841 gcgtgtcgtc agaccgtcta cgccgacctc gttcaacagg tccagggcgg cacggatcac
     3901 tgtattcggc tgcaactttg tcatgattga cactttatca ctgataaaca taatatgtcc
     3961 accaacttat cagtgataaa gaatccgcgc gttcaatcgg accagcggag gctggtccgg
     4021 aggccagacg tgaaacccaa catacccctg atcgtaattc tgagcactgt cgcgctcgac
     4081 gctgtcggca tcggcctgat tatgccggtg ctgccgggcc tcctgcgcga tctggttcac
     4141 tcgaacgacg tcaccgccca ctatggcatt ctgctggcgc tgtatgcgtt ggtgcaattt
     4201 gcctgcgcac ctgtgctggg cgcgctgtcg gatcgtttcg ggcggcggcc aatcttgctc
     4261 gtctcgctgg ccggcgccac tgtcgactac gccatcatgg cgacagcgcc tttcctttgg
     4321 gttctctata tcgggcggat cgtggccggc atcaccgggg cgactggggc ggtagccggc
     4381 gcttatattg ccgatatcac tgatggcgat gagcgcgcgc ggcacttcgg cttcatgagc
     4441 gcctgtttcg ggttcgggat ggtcgcggga cctgtgctcg gtgggctgat gggcggtttc
     4501 tccccccacg ctccgttctt cgccgcggca gccttgaacg gcctcaattt cctgacgggc
     4561 tgtttccttt tgccggagtc gcacaaaggc gaacgccggc cgttacgccg ggaggctctc
     4621 aacccgctcg cttcgttccg gtgggcccgg ggcatgaccg tcgtcgccgc cctgatggcg
     4681 gtcttcttca tcatgcaact tgtcggacag gtgccggccg cgctttgggt cattttcggc
     4741 gaggatcgct ttcactggga cgcgaccacg atcggcattt cgcttgccgc atttggcatt
     4801 ctgcattcac tcgcccaggc aatgatcacc ggccctgtag ccgcccggct cggcgaaagg
     4861 cgggcactca tgctcggaat gattgccgac ggcacaggct acatcctgct tgccttcgcg
     4921 acacggggat ggatggcgtt cccgatcatg gtcctgcttg cttcgggtgg catcggaatg
     4981 ccggcgctgc aagcaatgtt gtccaggcag gtggatgagg aacgtcaggg gcagctgcaa
     5041 ggctcactgg cggcgctcac cagcctgacc tcgatcgtcg gacccctcct cttcacggcg
     5101 atctatgcgg cttctataac aacgtggaac gggtgggcat ggattgcagg cgctgccctc
     5161 tacttgctct gcctgccggc gctgcgtcgc gggctttgga gcggcgcagg gcaacgagcc
     5221 gatcgctgat cgtggaaacg ataggcctat gccatgcggg tcaaggcgac ttccggcaag
     5281 ctatacgcgc cctagaattg tcaattttaa tcctctgttt atcggcagtt cgtagagcgc
     5341 gccgtgcgtc ccgagcgata ctgagcgaag caagtgcgtc gagcagtgcc cgcttgttcc
     5401 tgaaatgcca gtaaagcgct ggctgctgaa cccccagccg gaactgaccc cacaaggccc
     5461 tagcgtttgc aatgcaccag gtcatcattg acccaggcgt gttccaccag gccgctgcct
     5521 cgcaactctt cgcaggcttc gccgacctgc tcgcgccact tcttcacgcg ggtggaatcc
     5581 gatccgcaca tgaggcggaa ggtttccagc ttgagcgggt acggctcccg gtgcgagctg
     5641 aaatagtcga acatccgtcg ggccgtcggc gacagcttgc ggtacttctc ccatatgaat
     5701 ttcgtgtagt ggtcgccagc aaacagcacg acgatttcct cgtcgatcag gacctggcaa
     5761 cgggacgttt tcttgccacg gtccaggacg cggaagcggt gcagcagcga caccgattcc
     5821 aggtgcccaa cgcggtcgga cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat
     5881 tcctcggcct tcgtgtaata ccggccattg atcgaccagc ccaggtcctg gcaaagctcg
     5941 tagaacgtga aggtgatcgg ctcgccgata ggggtgcgct tcgcgtactc caacacctgc
     6001 tgccacacca gttcgtcatc gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg
     6061 tccttgttga cgtggaaaat gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg
     6121 cgcgtggtga acagggcaga gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc
     6181 cacggcgcaa tatcgaacaa ggaaagctgc atttccttga tctgctgctt cgtgtgtttc
     6241 agcaacgcgg cctgcttggc ctcgctgacc tgttttgcca ggtcctcgcc ggcggttttt
     6301 cgcttcttgg tcgtcatagt tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc
     6361 gcctcctgtt cgagacgacg cgaacgctcc acggcggccg atggcgcggg cagggcaggg
     6421 ggagccagtt gcacgctgtc gcgctcgatc ttggccgtag cttgctggac catcgagccg
     6481 acggactgga aggtttcgcg gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca
     6541 tcctcggcgg aaaaccccgc gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc
     6601 cgattcattc accctccttg cgggattgcc ccgactcacg ccggggcaat gtgcccttat
     6661 tcctgatttg acccgcctgg tgccttggtg tccagataat ccaccttatc ggcaatgaag
     6721 tcggtcccgt agaccgtctg gccgtccttc tcgtacttgg tattccgaat cttgccctgc
     6781 acgaatacca gctccgcgaa gtcgctcttc ttgatggagc gcatggggac gtgcttggca
     6841 atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt catggctctg ccctcgggcg
     6901 gaccacgccc atcatgacct tgccaagctc gtcctgcttc tcttcgatct tcgccagcag
     6961 ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga gccagagttt
     7021 cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc ggacgtgctc
     7081 atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt aggcctacag
     7141 gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg gaaggcagta
     7201 caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca tccgcttgcc
     7261 ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc cgttgagcac
     7321 cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc
     7381 acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg
     7441 gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac
     7501 cccgaagcag ggttatgcag cggaaaagat ccgtc
//
