LOCUS       sequence-69231-e        8180 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cumate-inducible expression of ECFP-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8180)
  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 8180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pQC
FEATURES             Location/Qualifiers
     source          1..8180
                     /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             join(2562..2564,2565..2567,2568..3278)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2607..2628)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2868..2887)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3215..3236
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3330..3395
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(3467..3484)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3467..3483)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3476..3498)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3692..3711)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3811..3833
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3871..3889)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4675..5874
                     /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(6105..7253)
                     /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(7525..7896)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7929..8038
                     /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 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2641 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2701 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2761 ggggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2821 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2881 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2941 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3001 tcagccacaa cgtctatatc accgccgaca agcagaagaa cggcatcaag gccaacttca
     3061 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3121 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3181 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3241 ccgccgggat cactctcggc atggacgagc tgtacaagat taatatgact ctagaagatc
     3301 tcgaggtacc gagctccaat tgggatccgg actacaagga ccacgacggc gactataagg
     3361 accacgacat cgactacaag gatgacgacg acaagtgagg atccggtgaa gtgacccgca
     3421 aaaaaaaagg ccaggggatc gctcccccgg cctttttttt gaattcactg gccgtcgttt
     3481 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3541 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3601 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     3661 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3721 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3781 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3841 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3901 atgtcatgat aataatggtt tcttagcacc ctttctcggt ccttcaacgt tcctgacaac
     3961 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
     4021 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
     4081 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
     4141 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
     4201 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
     4261 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
     4321 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
     4381 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
     4441 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
     4501 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
     4561 ctttgtcatg attgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
     4621 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
     4681 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
     4741 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
     4801 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
     4861 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
     4921 gccactgtcg actacgccat catggcgaca gcgcctttcc tttgggttct ctatatcggg
     4981 cggatcgtgg ccggcatcac cggggcgact ggggcggtag ccggcgctta tattgccgat
     5041 atcactgatg gcgatgagcg cgcgcggcac ttcggcttca tgagcgcctg tttcgggttc
     5101 gggatggtcg cgggacctgt gctcggtggg ctgatgggcg gtttctcccc ccacgctccg
     5161 ttcttcgccg cggcagcctt gaacggcctc aatttcctga cgggctgttt ccttttgccg
     5221 gagtcgcaca aaggcgaacg ccggccgtta cgccgggagg ctctcaaccc gctcgcttcg
     5281 ttccggtggg cccggggcat gaccgtcgtc gccgccctga tggcggtctt cttcatcatg
     5341 caacttgtcg gacaggtgcc ggccgcgctt tgggtcattt tcggcgagga tcgctttcac
     5401 tgggacgcga ccacgatcgg catttcgctt gccgcatttg gcattctgca ttcactcgcc
     5461 caggcaatga tcaccggccc tgtagccgcc cggctcggcg aaaggcgggc actcatgctc
     5521 ggaatgattg ccgacggcac aggctacatc ctgcttgcct tcgcgacacg gggatggatg
     5581 gcgttcccga tcatggtcct gcttgcttcg ggtggcatcg gaatgccggc gctgcaagca
     5641 atgttgtcca ggcaggtgga tgaggaacgt caggggcagc tgcaaggctc actggcggcg
     5701 ctcaccagcc tgacctcgat cgtcggaccc ctcctcttca cggcgatcta tgcggcttct
     5761 ataacaacgt ggaacgggtg ggcatggatt gcaggcgctg ccctctactt gctctgcctg
     5821 ccggcgctgc gtcgcgggct ttggagcggc gcagggcaac gagccgatcg ctgatcgtgg
     5881 aaacgatagg cctatgccat gcgggtcaag gcgacttccg gcaagctata cgcgccctag
     5941 aattgtcaat tttaatcctc tgtttatcgg cagttcgtag agcgcgccgt gcgtcccgag
     6001 cgatactgag cgaagcaagt gcgtcgagca gtgcccgctt gttcctgaaa tgccagtaaa
     6061 gcgctggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg tttgcaatgc
     6121 accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa ctcttcgcag
     6181 gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc gcacatgagg
     6241 cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata gtcgaacatc
     6301 cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt gtagtggtcg
     6361 ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga cgttttcttg
     6421 ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg cccaacgcgg
     6481 tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc ggccttcgtg
     6541 taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa cgtgaaggtg
     6601 atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca caccagttcg
     6661 tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt gttgacgtgg
     6721 aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt ggtgaacagg
     6781 gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg cgcaatatcg
     6841 aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa cgcggcctgc
     6901 ttggcctcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt cttggtcgtc
     6961 atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc ctgttcgaga
     7021 cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc cagttgcacg
     7081 ctgtcgcgct cgatcttggc cgtagcttgc tggaccatcg agccgacgga ctggaaggtt
     7141 tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc ggcggaaaac
     7201 cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt cattcaccct
     7261 ccttgcggga ttgccccgac tcacgccggg gcaatgtgcc cttattcctg atttgacccg
     7321 cctggtgcct tggtgtccag ataatccacc ttatcggcaa tgaagtcggt cccgtagacc
     7381 gtctggccgt ccttctcgta cttggtattc cgaatcttgc cctgcacgaa taccagctcc
     7441 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
     7501 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
     7561 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
     7621 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
     7681 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
     7741 cgtgattttg tagccctggc cgacggccag caggtaggcc tacaggctca tgccggccgc
     7801 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
     7861 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
     7921 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
     7981 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
     8041 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
     8101 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
     8161 tgcagcggaa aagatccgtc
//
