LOCUS       sequence-69227-e        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate-inducible expression of HA-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  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: pQH
FEATURES             Location/Qualifiers
     source          1..7493
                     /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..2591
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2643..2708
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2780..2797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2780..2796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2789..2811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3005..3024)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3124..3146
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3184..3202)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3988..5187
                     /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(5418..6566)
                     /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(6838..7209)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7242..7351
                     /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 catgtacccg tacgacgtcc
     2581 cggactacgc cattaatatg actctagaag atctcgaggt accgagctcc aattgggatc
     2641 cggactacaa ggaccacgac ggcgactata aggaccacga catcgactac aaggatgacg
     2701 acgacaagtg aggatccggt gaagtgaccc gcaaaaaaaa aggccagggg atcgctcccc
     2761 cggccttttt tttgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2821 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2881 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
     2941 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     3001 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3061 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3121 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3181 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttagc
     3241 accctttctc ggtccttcaa cgttcctgac aacgagcctc cttttcgcca atccatcgac
     3301 aatcaccgcg agtccctgct cgaacgctgc gtccggaccg gcttcgtcga aggcgtctat
     3361 cgcggcccgc aacagcggcg agagcggagc ctgttcaacg gtgccgccgc gctcgccggc
     3421 atcgctgtcg ccggcctgct cctcaagcac ggccccaaca gtgaagtagc tgattgtcat
     3481 cagcgcattg acggcgtccc cggccgaaaa acccgcctcg cagaggaagc gaagctgcgc
     3541 gtcggccgtt tccatctgcg gtgcgcccgg tcgcgtgccg gcatggatgc gcgcgccatc
     3601 gcggtaggcg agcagcgcct gcctgaagct gcgggcattc ccgatcagaa atgagcgcca
     3661 gtcgtcgtcg gctctcggca ccgaatgcgt atgattctcc gccagcatgg cttcggccag
     3721 tgcgtcgagc agcgcccgct tgttcctgaa gtgccagtaa agcgccggct gctgaacccc
     3781 caaccgttcc gccagtttgc gtgtcgtcag accgtctacg ccgacctcgt tcaacaggtc
     3841 cagggcggca cggatcactg tattcggctg caactttgtc atgattgaca ctttatcact
     3901 gataaacata atatgtccac caacttatca gtgataaaga atccgcgcgt tcaatcggac
     3961 cagcggaggc tggtccggag gccagacgtg aaacccaaca tacccctgat cgtaattctg
     4021 agcactgtcg cgctcgacgc tgtcggcatc ggcctgatta tgccggtgct gccgggcctc
     4081 ctgcgcgatc tggttcactc gaacgacgtc accgcccact atggcattct gctggcgctg
     4141 tatgcgttgg tgcaatttgc ctgcgcacct gtgctgggcg cgctgtcgga tcgtttcggg
     4201 cggcggccaa tcttgctcgt ctcgctggcc ggcgccactg tcgactacgc catcatggcg
     4261 acagcgcctt tcctttgggt tctctatatc gggcggatcg tggccggcat caccggggcg
     4321 actggggcgg tagccggcgc ttatattgcc gatatcactg atggcgatga gcgcgcgcgg
     4381 cacttcggct tcatgagcgc ctgtttcggg ttcgggatgg tcgcgggacc tgtgctcggt
     4441 gggctgatgg gcggtttctc cccccacgct ccgttcttcg ccgcggcagc cttgaacggc
     4501 ctcaatttcc tgacgggctg tttccttttg ccggagtcgc acaaaggcga acgccggccg
     4561 ttacgccggg aggctctcaa cccgctcgct tcgttccggt gggcccgggg catgaccgtc
     4621 gtcgccgccc tgatggcggt cttcttcatc atgcaacttg tcggacaggt gccggccgcg
     4681 ctttgggtca ttttcggcga ggatcgcttt cactgggacg cgaccacgat cggcatttcg
     4741 cttgccgcat ttggcattct gcattcactc gcccaggcaa tgatcaccgg ccctgtagcc
     4801 gcccggctcg gcgaaaggcg ggcactcatg ctcggaatga ttgccgacgg cacaggctac
     4861 atcctgcttg ccttcgcgac acggggatgg atggcgttcc cgatcatggt cctgcttgct
     4921 tcgggtggca tcggaatgcc ggcgctgcaa gcaatgttgt ccaggcaggt ggatgaggaa
     4981 cgtcaggggc agctgcaagg ctcactggcg gcgctcacca gcctgacctc gatcgtcgga
     5041 cccctcctct tcacggcgat ctatgcggct tctataacaa cgtggaacgg gtgggcatgg
     5101 attgcaggcg ctgccctcta cttgctctgc ctgccggcgc tgcgtcgcgg gctttggagc
     5161 ggcgcagggc aacgagccga tcgctgatcg tggaaacgat aggcctatgc catgcgggtc
     5221 aaggcgactt ccggcaagct atacgcgccc tagaattgtc aattttaatc ctctgtttat
     5281 cggcagttcg tagagcgcgc cgtgcgtccc gagcgatact gagcgaagca agtgcgtcga
     5341 gcagtgcccg cttgttcctg aaatgccagt aaagcgctgg ctgctgaacc cccagccgga
     5401 actgacccca caaggcccta gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt
     5461 tccaccaggc cgctgcctcg caactcttcg caggcttcgc cgacctgctc gcgccacttc
     5521 ttcacgcggg tggaatccga tccgcacatg aggcggaagg tttccagctt gagcgggtac
     5581 ggctcccggt gcgagctgaa atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg
     5641 tacttctccc atatgaattt cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg
     5701 tcgatcagga cctggcaacg ggacgttttc ttgccacggt ccaggacgcg gaagcggtgc
     5761 agcagcgaca ccgattccag gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc
     5821 tgtaggcgcg acaggcattc ctcggccttc gtgtaatacc ggccattgat cgaccagccc
     5881 aggtcctggc aaagctcgta gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc
     5941 gcgtactcca acacctgctg ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg
     6001 gtgtaggtga tcttcacgtc cttgttgacg tggaaaatga ccttgttttg cagcgcctcg
     6061 cgcgggattt tcttgttgcg cgtggtgaac agggcagagc gggccgtgtc gtttggcatc
     6121 gctcgcatcg tgtccggcca cggcgcaata tcgaacaagg aaagctgcat ttccttgatc
     6181 tgctgcttcg tgtgtttcag caacgcggcc tgcttggcct cgctgacctg ttttgccagg
     6241 tcctcgccgg cggtttttcg cttcttggtc gtcatagttc ctcgcgtgtc gatggtcatc
     6301 gacttcgcca aacctgccgc ctcctgttcg agacgacgcg aacgctccac ggcggccgat
     6361 ggcgcgggca gggcaggggg agccagttgc acgctgtcgc gctcgatctt ggccgtagct
     6421 tgctggacca tcgagccgac ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg
     6481 cttgcgatgg tttcggcatc ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat
     6541 gccttccggt caaacgtccg attcattcac cctccttgcg ggattgcccc gactcacgcc
     6601 ggggcaatgt gcccttattc ctgatttgac ccgcctggtg ccttggtgtc cagataatcc
     6661 accttatcgg caatgaagtc ggtcccgtag accgtctggc cgtccttctc gtacttggta
     6721 ttccgaatct tgccctgcac gaataccagc tccgcgaagt cgctcttctt gatggagcgc
     6781 atggggacgt gcttggcaat cacgcgcacc ccccggccgt tttagcggct aaaaaagtca
     6841 tggctctgcc ctcgggcgga ccacgcccat catgaccttg ccaagctcgt cctgcttctc
     6901 ttcgatcttc gccagcaggg cgaggatcgt ggcatcaccg aaccgcgccg tgcgcgggtc
     6961 gtcggtgagc cagagtttca gcaggccgcc caggcggccc aggtcgccat tgatgcgggc
     7021 cagctcgcgg acgtgctcat agtccacgac gcccgtgatt ttgtagccct ggccgacggc
     7081 cagcaggtag gcctacaggc tcatgccggc cgccgccgcc ttttcctcaa tcgctcttcg
     7141 ttcgtctgga aggcagtaca ccttgatagg tgggctgccc ttcctggttg gcttggtttc
     7201 atcagccatc cgcttgccct catctgttac gccggcggta gccggccagc ctcgcagagc
     7261 aggattcccg ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg aacacccgct
     7321 cgcgggtggg cctacttcac ctatcctgcc cggctgacgc cgttggatac accaaggaaa
     7381 gtctacacga accctttggc aaaatcctgt atatcgtgcg aaaaaggatg gatataccga
     7441 aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg gaaaagatcc gtc
//
