LOCUS       sequence-69230-e        8171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate-inducible expression of mCherry-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8171)
  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 8171)
  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: pQR
FEATURES             Location/Qualifiers
     source          1..8171
                     /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             2562..3269
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2707..2725)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2975..2994
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3321..3386
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3458..3475)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3458..3474)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3467..3489)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3683..3702)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3802..3824
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3862..3880)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4666..5865
                     /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(6096..7244)
                     /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(7516..7887)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7920..8029
                     /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 catggtgagc aagggcgagg
     2581 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     2641 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     2701 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     2761 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     2821 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     2881 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     2941 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     3001 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     3061 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     3121 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     3181 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     3241 ccaccggcgg catggacgag ctgtacaaga ttaatatgac tctagaagat ctcgaggtac
     3301 cgagctccaa ttgggatccg gactacaagg accacgacgg cgactataag gaccacgaca
     3361 tcgactacaa ggatgacgac gacaagtgag gatccggtga agtgacccgc aaaaaaaaag
     3421 gccaggggat cgctcccccg gccttttttt tgaattcact ggccgtcgtt ttacaacgtc
     3481 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3541 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3601 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3661 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3721 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3781 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3841 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     3901 taataatggt ttcttagcac cctttctcgg tccttcaacg ttcctgacaa cgagcctcct
     3961 tttcgccaat ccatcgacaa tcaccgcgag tccctgctcg aacgctgcgt ccggaccggc
     4021 ttcgtcgaag gcgtctatcg cggcccgcaa cagcggcgag agcggagcct gttcaacggt
     4081 gccgccgcgc tcgccggcat cgctgtcgcc ggcctgctcc tcaagcacgg ccccaacagt
     4141 gaagtagctg attgtcatca gcgcattgac ggcgtccccg gccgaaaaac ccgcctcgca
     4201 gaggaagcga agctgcgcgt cggccgtttc catctgcggt gcgcccggtc gcgtgccggc
     4261 atggatgcgc gcgccatcgc ggtaggcgag cagcgcctgc ctgaagctgc gggcattccc
     4321 gatcagaaat gagcgccagt cgtcgtcggc tctcggcacc gaatgcgtat gattctccgc
     4381 cagcatggct tcggccagtg cgtcgagcag cgcccgcttg ttcctgaagt gccagtaaag
     4441 cgccggctgc tgaaccccca accgttccgc cagtttgcgt gtcgtcagac cgtctacgcc
     4501 gacctcgttc aacaggtcca gggcggcacg gatcactgta ttcggctgca actttgtcat
     4561 gattgacact ttatcactga taaacataat atgtccacca acttatcagt gataaagaat
     4621 ccgcgcgttc aatcggacca gcggaggctg gtccggaggc cagacgtgaa acccaacata
     4681 cccctgatcg taattctgag cactgtcgcg ctcgacgctg tcggcatcgg cctgattatg
     4741 ccggtgctgc cgggcctcct gcgcgatctg gttcactcga acgacgtcac cgcccactat
     4801 ggcattctgc tggcgctgta tgcgttggtg caatttgcct gcgcacctgt gctgggcgcg
     4861 ctgtcggatc gtttcgggcg gcggccaatc ttgctcgtct cgctggccgg cgccactgtc
     4921 gactacgcca tcatggcgac agcgcctttc ctttgggttc tctatatcgg gcggatcgtg
     4981 gccggcatca ccggggcgac tggggcggta gccggcgctt atattgccga tatcactgat
     5041 ggcgatgagc gcgcgcggca cttcggcttc atgagcgcct gtttcgggtt cgggatggtc
     5101 gcgggacctg tgctcggtgg gctgatgggc ggtttctccc cccacgctcc gttcttcgcc
     5161 gcggcagcct tgaacggcct caatttcctg acgggctgtt tccttttgcc ggagtcgcac
     5221 aaaggcgaac gccggccgtt acgccgggag gctctcaacc cgctcgcttc gttccggtgg
     5281 gcccggggca tgaccgtcgt cgccgccctg atggcggtct tcttcatcat gcaacttgtc
     5341 ggacaggtgc cggccgcgct ttgggtcatt ttcggcgagg atcgctttca ctgggacgcg
     5401 accacgatcg gcatttcgct tgccgcattt ggcattctgc attcactcgc ccaggcaatg
     5461 atcaccggcc ctgtagccgc ccggctcggc gaaaggcggg cactcatgct cggaatgatt
     5521 gccgacggca caggctacat cctgcttgcc ttcgcgacac ggggatggat ggcgttcccg
     5581 atcatggtcc tgcttgcttc gggtggcatc ggaatgccgg cgctgcaagc aatgttgtcc
     5641 aggcaggtgg atgaggaacg tcaggggcag ctgcaaggct cactggcggc gctcaccagc
     5701 ctgacctcga tcgtcggacc cctcctcttc acggcgatct atgcggcttc tataacaacg
     5761 tggaacgggt gggcatggat tgcaggcgct gccctctact tgctctgcct gccggcgctg
     5821 cgtcgcgggc tttggagcgg cgcagggcaa cgagccgatc gctgatcgtg gaaacgatag
     5881 gcctatgcca tgcgggtcaa ggcgacttcc ggcaagctat acgcgcccta gaattgtcaa
     5941 ttttaatcct ctgtttatcg gcagttcgta gagcgcgccg tgcgtcccga gcgatactga
     6001 gcgaagcaag tgcgtcgagc agtgcccgct tgttcctgaa atgccagtaa agcgctggct
     6061 gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg caccaggtca
     6121 tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca ggcttcgccg
     6181 acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt
     6241 tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc
     6301 gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc gccagcaaac
     6361 agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt gccacggtcc
     6421 aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg gtcggacgtg
     6481 aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt gtaataccgg
     6541 ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg
     6601 ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc gtcatcgtcg
     6661 gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc
     6721 ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg
     6781 gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa
     6841 agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg
     6901 ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt catagttcct
     6961 cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa
     7021 cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc
     7081 tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt ttcgcggggc
     7141 gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg
     7201 atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc tccttgcggg
     7261 attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc
     7321 ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac cgtctggccg
     7381 tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagctc cgcgaagtcg
     7441 ctcttcttga tggagcgcat ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt
     7501 tagcggctaa aaaagtcatg gctctgccct cgggcggacc acgcccatca tgaccttgcc
     7561 aagctcgtcc tgcttctctt cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa
     7621 ccgcgccgtg cgcgggtcgt cggtgagcca gagtttcagc aggccgccca ggcggcccag
     7681 gtcgccattg atgcgggcca gctcgcggac gtgctcatag tccacgacgc ccgtgatttt
     7741 gtagccctgg ccgacggcca gcaggtaggc ctacaggctc atgccggccg ccgccgcctt
     7801 ttcctcaatc gctcttcgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt
     7861 cctggttggc ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtagc
     7921 cggccagcct cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat aagggacagt
     7981 gaagaaggaa cacccgctcg cgggtgggcc tacttcacct atcctgcccg gctgacgccg
     8041 ttggatacac caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa
     8101 aaaggatgga tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga
     8161 aaagatccgt c
//
