LOCUS       sequence-69228-e        7502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate-inducible expression of myc-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7502)
  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 7502)
  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: pQM
FEATURES             Location/Qualifiers
     source          1..7502
                     /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..2594
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2652..2717
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2789..2806)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2789..2805)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2798..2820)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3014..3033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3133..3155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3193..3211)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3997..5196
                     /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(5427..6575)
                     /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(6847..7218)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7251..7360
                     /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 catggagcag aagctgatct
     2581 cggaagagga cctgggctcg attaatatga ctctagaaga tctcgaggta ccgagctcca
     2641 attgggatcc ggactacaag gaccacgacg gcgactataa ggaccacgac atcgactaca
     2701 aggatgacga cgacaagtga ggatccggtg aagtgacccg caaaaaaaaa ggccagggga
     2761 tcgctccccc ggcctttttt ttgaattcac tggccgtcgt tttacaacgt cgtgactggg
     2821 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2881 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2941 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     3001 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     3061 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3121 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3181 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     3241 tttcttagca ccctttctcg gtccttcaac gttcctgaca acgagcctcc ttttcgccaa
     3301 tccatcgaca atcaccgcga gtccctgctc gaacgctgcg tccggaccgg cttcgtcgaa
     3361 ggcgtctatc gcggcccgca acagcggcga gagcggagcc tgttcaacgg tgccgccgcg
     3421 ctcgccggca tcgctgtcgc cggcctgctc ctcaagcacg gccccaacag tgaagtagct
     3481 gattgtcatc agcgcattga cggcgtcccc ggccgaaaaa cccgcctcgc agaggaagcg
     3541 aagctgcgcg tcggccgttt ccatctgcgg tgcgcccggt cgcgtgccgg catggatgcg
     3601 cgcgccatcg cggtaggcga gcagcgcctg cctgaagctg cgggcattcc cgatcagaaa
     3661 tgagcgccag tcgtcgtcgg ctctcggcac cgaatgcgta tgattctccg ccagcatggc
     3721 ttcggccagt gcgtcgagca gcgcccgctt gttcctgaag tgccagtaaa gcgccggctg
     3781 ctgaaccccc aaccgttccg ccagtttgcg tgtcgtcaga ccgtctacgc cgacctcgtt
     3841 caacaggtcc agggcggcac ggatcactgt attcggctgc aactttgtca tgattgacac
     3901 tttatcactg ataaacataa tatgtccacc aacttatcag tgataaagaa tccgcgcgtt
     3961 caatcggacc agcggaggct ggtccggagg ccagacgtga aacccaacat acccctgatc
     4021 gtaattctga gcactgtcgc gctcgacgct gtcggcatcg gcctgattat gccggtgctg
     4081 ccgggcctcc tgcgcgatct ggttcactcg aacgacgtca ccgcccacta tggcattctg
     4141 ctggcgctgt atgcgttggt gcaatttgcc tgcgcacctg tgctgggcgc gctgtcggat
     4201 cgtttcgggc ggcggccaat cttgctcgtc tcgctggccg gcgccactgt cgactacgcc
     4261 atcatggcga cagcgccttt cctttgggtt ctctatatcg ggcggatcgt ggccggcatc
     4321 accggggcga ctggggcggt agccggcgct tatattgccg atatcactga tggcgatgag
     4381 cgcgcgcggc acttcggctt catgagcgcc tgtttcgggt tcgggatggt cgcgggacct
     4441 gtgctcggtg ggctgatggg cggtttctcc ccccacgctc cgttcttcgc cgcggcagcc
     4501 ttgaacggcc tcaatttcct gacgggctgt ttccttttgc cggagtcgca caaaggcgaa
     4561 cgccggccgt tacgccggga ggctctcaac ccgctcgctt cgttccggtg ggcccggggc
     4621 atgaccgtcg tcgccgccct gatggcggtc ttcttcatca tgcaacttgt cggacaggtg
     4681 ccggccgcgc tttgggtcat tttcggcgag gatcgctttc actgggacgc gaccacgatc
     4741 ggcatttcgc ttgccgcatt tggcattctg cattcactcg cccaggcaat gatcaccggc
     4801 cctgtagccg cccggctcgg cgaaaggcgg gcactcatgc tcggaatgat tgccgacggc
     4861 acaggctaca tcctgcttgc cttcgcgaca cggggatgga tggcgttccc gatcatggtc
     4921 ctgcttgctt cgggtggcat cggaatgccg gcgctgcaag caatgttgtc caggcaggtg
     4981 gatgaggaac gtcaggggca gctgcaaggc tcactggcgg cgctcaccag cctgacctcg
     5041 atcgtcggac ccctcctctt cacggcgatc tatgcggctt ctataacaac gtggaacggg
     5101 tgggcatgga ttgcaggcgc tgccctctac ttgctctgcc tgccggcgct gcgtcgcggg
     5161 ctttggagcg gcgcagggca acgagccgat cgctgatcgt ggaaacgata ggcctatgcc
     5221 atgcgggtca aggcgacttc cggcaagcta tacgcgccct agaattgtca attttaatcc
     5281 tctgtttatc ggcagttcgt agagcgcgcc gtgcgtcccg agcgatactg agcgaagcaa
     5341 gtgcgtcgag cagtgcccgc ttgttcctga aatgccagta aagcgctggc tgctgaaccc
     5401 ccagccggaa ctgaccccac aaggccctag cgtttgcaat gcaccaggtc atcattgacc
     5461 caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc aggcttcgcc gacctgctcg
     5521 cgccacttct tcacgcgggt ggaatccgat ccgcacatga ggcggaaggt ttccagcttg
     5581 agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca tccgtcgggc cgtcggcgac
     5641 agcttgcggt acttctccca tatgaatttc gtgtagtggt cgccagcaaa cagcacgacg
     5701 atttcctcgt cgatcaggac ctggcaacgg gacgttttct tgccacggtc caggacgcgg
     5761 aagcggtgca gcagcgacac cgattccagg tgcccaacgc ggtcggacgt gaagcccatc
     5821 gccgtcgcct gtaggcgcga caggcattcc tcggccttcg tgtaataccg gccattgatc
     5881 gaccagccca ggtcctggca aagctcgtag aacgtgaagg tgatcggctc gccgataggg
     5941 gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt cgtcatcgtc ggcccgcagc
     6001 tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt ggaaaatgac cttgttttgc
     6061 agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca gggcagagcg ggccgtgtcg
     6121 tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat cgaacaagga aagctgcatt
     6181 tccttgatct gctgcttcgt gtgtttcagc aacgcggcct gcttggcctc gctgacctgt
     6241 tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg tcatagttcc tcgcgtgtcg
     6301 atggtcatcg acttcgccaa acctgccgcc tcctgttcga gacgacgcga acgctccacg
     6361 gcggccgatg gcgcgggcag ggcaggggga gccagttgca cgctgtcgcg ctcgatcttg
     6421 gccgtagctt gctggaccat cgagccgacg gactggaagg tttcgcgggg cgcacgcatg
     6481 acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa accccgcgtc gatcagttct
     6541 tgcctgtatg ccttccggtc aaacgtccga ttcattcacc ctccttgcgg gattgccccg
     6601 actcacgccg gggcaatgtg cccttattcc tgatttgacc cgcctggtgc cttggtgtcc
     6661 agataatcca ccttatcggc aatgaagtcg gtcccgtaga ccgtctggcc gtccttctcg
     6721 tacttggtat tccgaatctt gccctgcacg aataccagct ccgcgaagtc gctcttcttg
     6781 atggagcgca tggggacgtg cttggcaatc acgcgcaccc cccggccgtt ttagcggcta
     6841 aaaaagtcat ggctctgccc tcgggcggac cacgcccatc atgaccttgc caagctcgtc
     6901 ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga accgcgccgt
     6961 gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca ggtcgccatt
     7021 gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg
     7081 gccgacggcc agcaggtagg cctacaggct catgccggcc gccgccgcct tttcctcaat
     7141 cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg
     7201 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
     7261 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     7321 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     7381 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     7441 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaagatccg
     7501 tc
//
