LOCUS       sequence-211919-        7783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescence-based gauging of the level of the nucleotide secondary 
            messenger cyclic di-GMP in Pseudomonas aeruginosa and related 
            species. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7783)
  AUTHORS   Rybtke MT, Borlee BR, Murakami K, Irie Y, Hentzer M, Nielsen TE, 
            Givskov M, Parsek MR, Tolker-Nielsen T
  TITLE     Fluorescence-based reporter for gauging cyclic di-GMP levels in 
            Pseudomonas aeruginosa.
  JOURNAL   Appl Environ Microbiol. 2012 Aug;78(15):5060-9. doi: 
            10.1128/AEM.00414-12. Epub 2012 May 11.
  PUBMED    22582064
REFERENCE   2  (bases 1 to 7783)
  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.00414-12"; journalName: "Appl Environ Microbiol"; date:
            "2012-08"; volume: "78"; issue: "15"; pages: "5060-9"
COMMENT     Sequence Label: pTn7CdrA-gfp(ASV)C
FEATURES             Location/Qualifiers
     source          1..7783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..38)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(192..780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(272..291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(951..1742,1743..1811))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1574..1593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1812..1916)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1984..2002
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2040..2062)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2162..2181
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2375..2397
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2389..2406
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2390..2406
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     mobile_element  2451..2616
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        join(2740..2745,2746..2762,2763..2768)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2957..3490
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     RBS             3919..3930
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3943..4656
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             3943..4656
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3973..4001)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4596..4616
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4721..4815
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4859..5518
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4925..4944)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5583..5669
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     oriT            6623..6732
                     /note="incP origin of transfer"
     CDS             6765..7136
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
ORIGIN
        1 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
       61 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
      121 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      181 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      241 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      301 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      361 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      421 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      481 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      541 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      601 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
      661 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
      721 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
      781 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
      841 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      901 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      961 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1021 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1081 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1141 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1201 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1261 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1321 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1381 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     1441 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1501 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1561 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     1621 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     1681 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     1741 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1801 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1861 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1921 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1981 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     2041 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2101 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     2161 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     2221 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     2281 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     2341 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     2401 gccagtgaat tcgagctcgc ccaaacataa caggaagaaa aatgccccgc tgtgggcgga
     2461 caaaatagtt gggaactggg aggggtggaa atggagtttt taaggattat ttagggaaga
     2521 gtgacaaaat agatgggaac tgggtgtagc gtcgtaagct aatacgaaaa ttaaaaatga
     2581 caaaatagtt tggaactaga tttcacttat ctggttggcc tgcaggtcga ctagagtcgg
     2641 ggatccggtg attgattgag caagctttat gcttgtaaac cgttttgtga aaaaattttt
     2701 aaaataaaaa aggggacctc tagggtcccc aattacgaat tgacataagc ctgttcggtt
     2761 cgtaaactgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     2821 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttgta
     2881 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
     2941 ttatggagca gcaacgatgt tacgcagcag caacgatgtt acgcagcagg gcagtcgccc
     3001 taaaacaaag ttaggtggct caagtatggg catcattcgc acatgtaggc tcggccctga
     3061 ccaagtcaaa tccatgcggg ctgctcttga tcttttcggt cgtgagttcg gagacgtagc
     3121 cacctactcc caacatcagc cggactccga ttacctcggg aacttgctcc gtagtaagac
     3181 attcatcgcg cttgctgcct tcgaccaaga agcggttgtt ggcgctctcg cggcttacgt
     3241 tctgcccaag tttgagcagc cgcgtagtga gatctatatc tatgatctcg cagtctccgg
     3301 cgagcaccgg aggcagggca ttgccaccgc gctcatcaat ctcctcaagc atgaggccaa
     3361 cgcgcttggt gcttatgtga tctacgtgca agcagattac ggtgacgatc ccgcagtggc
     3421 tctctataca aagttgggca tacgggaaga agtgatgcac tttgatatcg acccaagtac
     3481 cgccacctaa caattcgttc aagccgagat cggcttcccg gccgcggagt tgttcggtaa
     3541 attgtcacaa cgccgcggcc aattctaatt ggggacccta gaggtcccct tttttatttt
     3601 aaaaattttt tcacaaaacg gtttacaagc ataaagcttg ctcaatcaat caccggatcc
     3661 cgacgggccc ggtaccgcgg ccgcgcgaat tcgagctcgg tacccgggga tcctctagac
     3721 attgtcggtt ttttgacggt attacatatt taacctccgt agtgtcattt ttgagtcact
     3781 ccgacgaacg gtcgcccttt caagtggaca atttttgatc tgaaaggcat ttttttggca
     3841 cacaaggcgc aatgtgaatt gcttctcaga ttttcgtcat ttaactgacg aaatgcagtg
     3901 ctggaaaact gacgtgcgaa agaggagaaa ttaactatga gcatgcgtaa aggagaagaa
     3961 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     4021 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
     4081 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttcggttat
     4141 ggtgttcaat gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt
     4201 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     4261 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     4321 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac
     4381 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa
     4441 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact
     4501 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc
     4561 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct
     4621 gctgggatta cacatggcat ggatgaacta tacaaaaggc ctgcagcaaa cgacgaaaac
     4681 tacgctgcat cagtttaata agcttaatta gctgagcttg gactcctgtt gatagatcca
     4741 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     4801 tttattggtg agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat
     4861 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     4921 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     4981 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     5041 cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga
     5101 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     5161 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     5221 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     5281 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     5341 caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga
     5401 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     5461 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt
     5521 tttttaaggc agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc
     5581 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     5641 cggtgaacgc tctcctgagt aggacaaatc cgccgctagg agcttgcggc cgccttaaga
     5701 cgcgtggatc cccgggaatt tcgaccccac gcccctcttt aatacgacgg gcaatttgca
     5761 cttcagaaaa tgaagagttt gctttagcca taacaaaagt ccagtatgct ttttcacagc
     5821 ataactggac tgatttcagt ttacaactat tctgtctagt ttaagacttt attgtcatag
     5881 tttagatcta ttttgttcag tttaagactt tattgtccgc ccacacccgc ttacgcaggg
     5941 catccattta ttactcaacc gtaaccgatt ttgccaggtt acgcggctgg tcctctagag
     6001 gatccttttt gtccggtgtt gggttgaagg tgaagccggt cggggccgca gcgggggccg
     6061 gcttttcagc cttgcccccc tgcttcggcc gccgtggctc cggcgtcttg ggtgccggcg
     6121 cgggttccgc agccttggcc tgcggtgcgg gcacatcggc gggcttggcc ttgatgtgcc
     6181 gcctggcgtg cgagcggaac gtctcgtagg agaacttgac cttccccgtt tcccgcatgt
     6241 gctcccaaat ggtgacgagc gcatagccgg acgctaacgc cgcctcgaca tccgccctca
     6301 ccgccaggaa cgcaaccgca gcctcatcac gccggcgctt cttggccgcg cgggattcaa
     6361 cccactcggc cagctcgtcg gtgtagctct ttggcatcgt ctctcgcctg tcccctcagt
     6421 tcagtaattt cctgcatttg cctgtttcca gtcggtagat attccacaaa acagcaggga
     6481 agcagcgctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat
     6541 atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt
     6601 ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt
     6661 gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc
     6721 gaggctggcc ggctaccgcc ggcgtaacag atgagggcaa gcggatggct gatgaaacca
     6781 agccaaccag gaagggcagc ccacctatca aggtgtactg ccttccagac gaacgaagag
     6841 cgattgagga aaaggcggcg gcggccggca tgagcctgtc ggcctacctg ctggccgtcg
     6901 gccagggcta caaaatcacg ggcgtcgtgg actatgagca cgtccgcgag ctggcccgca
     6961 tcaatggcga cctgggccgc ctgggcggcc tgctgaaact ctggctcacc gacgacccgc
     7021 gcacggcgcg gttcggtgat gccacgatcc tcgccctgct ggcgaagatc gaagagaagc
     7081 aggacgagct tggcaaggtc atgatgggcg tggtccgccc gagggcagag ccatgacttt
     7141 tttagccgct aaaacggccg gggggtgcgc gtgattgcca agcacgtccc catgcgctcc
     7201 atcaagaaga gcgacttcgc ggagctggtg aagtacatca ccgacgagca aggcaagacc
     7261 gagcgcctgg gtcacgtgcg cgtcacgaac tgcgaggcaa acaccctgcc cgctgtcatg
     7321 gccgaggtga tggcgaccca gcacggcaac acccgttccg aggccgacaa gacctatcac
     7381 ctgctggtta gcttccgcgc gggagagaag cccgacgcgg agacgttgcg cgcgattgag
     7441 gaccgcatct gcgctgggct tggcttcgcc gagcatcagc gcgtcagtgc cgtgcatcac
     7501 gacaccgaca acctgcacat ccatatcgcc atcaacaaga ttcacccgac ccgaaacacc
     7561 atccatgagc cgtatcgggc ctaccgcgcc ctcgctgacc tctgcgcgac gctcgaacgg
     7621 gactacgggc ttgagcgtga caatcacgaa acgcggcagc gcgtttccga gaaccgcgcg
     7681 aacgacatgg agcggcacgc gggcgtggaa agcctggtcg gctggatcct ctagagtcct
     7741 gcattaatga atcggccaac gcgcggggag aggcggtttg cgt
//
