LOCUS       sequence-211915-        7741 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 7741)
  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 7741)
  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-gfpC
FEATURES             Location/Qualifiers
     source          1..7741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..81)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(235..823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(315..334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(994..1785,1786..1854))
                     /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     1617..1636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1855..1959)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2027..2045
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2083..2105)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2205..2224
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2418..2440
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2432..2449
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2433..2449
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     mobile_element  2494..2659
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     promoter        join(2783..2788,2789..2805,2806..2811)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3000..3533
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     RBS             3962..3973
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3986..4699
                     /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"
     primer_bind     complement(4016..4044)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4639..4659
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4722..4816
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4860..5519
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4926..4945)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5584..5670
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     oriT            6624..6733
                     /note="incP origin of transfer"
     CDS             6766..7137
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
ORIGIN
        1 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
       61 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      121 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      181 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      241 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      301 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      361 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      421 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      481 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      541 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      601 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      661 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      721 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      781 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      841 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
      901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
      961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     1801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     1861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1921 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1981 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     2041 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2101 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2161 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     2221 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     2281 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2341 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2401 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattcgagct
     2461 cgcccaaaca taacaggaag aaaaatgccc cgctgtgggc ggacaaaata gttgggaact
     2521 gggaggggtg gaaatggagt ttttaaggat tatttaggga agagtgacaa aatagatggg
     2581 aactgggtgt agcgtcgtaa gctaatacga aaattaaaaa tgacaaaata gtttggaact
     2641 agatttcact tatctggttg gcctgcaggt cgactagagt cggggatccg gtgattgatt
     2701 gagcaagctt tatgcttgta aaccgttttg tgaaaaaatt tttaaaataa aaaaggggac
     2761 ctctagggtc cccaattacg aattgacata agcctgttcg gttcgtaaac tgtaatgcaa
     2821 gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg
     2881 cgcagtggcg gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc
     2941 atccaagcag caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
     3001 tgttacgcag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg
     3061 gctcaagtat gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc
     3121 gggctgctct tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc
     3181 agccggactc cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg
     3241 ccttcgacca agaagcggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc
     3301 agccgcgtag tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg
     3361 gcattgccac cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg
     3421 tgatctacgt gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg
     3481 gcatacggga agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg
     3541 ttcaagccga gatcggcttc ccggccgcgg agttgttcgg taaattgtca caacgccgcg
     3601 gccaattcta attggggacc ctagaggtcc ccttttttat tttaaaaatt ttttcacaaa
     3661 acggtttaca agcataaagc ttgctcaatc aatcaccgga tcccgacggg cccggtaccg
     3721 cggccgcgcg aattcgagct cggtacccgg ggatcctcta gacattgtcg gttttttgac
     3781 ggtattacat atttaacctc cgtagtgtca tttttgagtc actccgacga acggtcgccc
     3841 tttcaagtgg acaatttttg atctgaaagg catttttttg gcacacaagg cgcaatgtga
     3901 attgcttctc agattttcgt catttaactg acgaaatgca gtgctggaaa actgacgtgc
     3961 gaaagaggag aaattaacta tgagcatgcg taaaggagaa gaacttttca ctggagttgt
     4021 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
     4081 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
     4141 actacctgtt ccatggccaa cacttgtcac tactttcggt tatggtgttc aatgctttgc
     4201 gagataccca gatcatatga aacagcatga ctttttcaag agtgccatgc ccgaaggtta
     4261 tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt
     4321 caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga
     4381 agatggaaac attcttggac acaaattgga atacaactat aactcacaca atgtatacat
     4441 catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga
     4501 agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
     4561 tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa
     4621 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
     4681 catggatgaa ctatacaaat aagcttaatt agctgagctt ggactcctgt tgatagatcc
     4741 agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt
     4801 ttttattggt gagaatccaa gctagcttgg cgagattttc aggagctaag gaagctaaaa
     4861 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
     4921 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
     4981 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
     5041 acattcttgc ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg
     5101 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     5161 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     5221 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     5281 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     5341 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     5401 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     5461 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     5521 ttttttaagg cagttattgg tgcccttaaa cgcctggggt aatgactctc tagcttgagg
     5581 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     5641 tcggtgaacg ctctcctgag taggacaaat ccgccgctag gagcttgcgg ccgccttaag
     5701 acgcgtggat ccccgggaat ttcgacccca cgcccctctt taatacgacg ggcaatttgc
     5761 acttcagaaa atgaagagtt tgctttagcc ataacaaaag tccagtatgc tttttcacag
     5821 cataactgga ctgatttcag tttacaacta ttctgtctag tttaagactt tattgtcata
     5881 gtttagatct attttgttca gtttaagact ttattgtccg cccacacccg cttacgcagg
     5941 gcatccattt attactcaac cgtaaccgat tttgccaggt tacgcggctg gtcctctaga
     6001 ggatcctttt tgtccggtgt tgggttgaag gtgaagccgg tcggggccgc agcgggggcc
     6061 ggcttttcag ccttgccccc ctgcttcggc cgccgtggct ccggcgtctt gggtgccggc
     6121 gcgggttccg cagccttggc ctgcggtgcg ggcacatcgg cgggcttggc cttgatgtgc
     6181 cgcctggcgt gcgagcggaa cgtctcgtag gagaacttga ccttccccgt ttcccgcatg
     6241 tgctcccaaa tggtgacgag cgcatagccg gacgctaacg ccgcctcgac atccgccctc
     6301 accgccagga acgcaaccgc agcctcatca cgccggcgct tcttggccgc gcgggattca
     6361 acccactcgg ccagctcgtc ggtgtagctc tttggcatcg tctctcgcct gtcccctcag
     6421 ttcagtaatt tcctgcattt gcctgtttcc agtcggtaga tattccacaa aacagcaggg
     6481 aagcagcgct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     6541 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     6601 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     6661 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     6721 cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc tgatgaaacc
     6781 aagccaacca ggaagggcag cccacctatc aaggtgtact gccttccaga cgaacgaaga
     6841 gcgattgagg aaaaggcggc ggcggccggc atgagcctgt cggcctacct gctggccgtc
     6901 ggccagggct acaaaatcac gggcgtcgtg gactatgagc acgtccgcga gctggcccgc
     6961 atcaatggcg acctgggccg cctgggcggc ctgctgaaac tctggctcac cgacgacccg
     7021 cgcacggcgc ggttcggtga tgccacgatc ctcgccctgc tggcgaagat cgaagagaag
     7081 caggacgagc ttggcaaggt catgatgggc gtggtccgcc cgagggcaga gccatgactt
     7141 ttttagccgc taaaacggcc ggggggtgcg cgtgattgcc aagcacgtcc ccatgcgctc
     7201 catcaagaag agcgacttcg cggagctggt gaagtacatc accgacgagc aaggcaagac
     7261 cgagcgcctg ggtcacgtgc gcgtcacgaa ctgcgaggca aacaccctgc ccgctgtcat
     7321 ggccgaggtg atggcgaccc agcacggcaa cacccgttcc gaggccgaca agacctatca
     7381 cctgctggtt agcttccgcg cgggagagaa gcccgacgcg gagacgttgc gcgcgattga
     7441 ggaccgcatc tgcgctgggc ttggcttcgc cgagcatcag cgcgtcagtg ccgtgcatca
     7501 cgacaccgac aacctgcaca tccatatcgc catcaacaag attcacccga cccgaaacac
     7561 catccatgag ccgtatcggg cctaccgcgc cctcgctgac ctctgcgcga cgctcgaacg
     7621 ggactacggg cttgagcgtg acaatcacga aacgcggcag cgcgtttccg agaaccgcgc
     7681 gaacgacatg gagcggcacg cgggcgtgga aagcctggtc ggctggatcc tctagagtcc
     7741 t
//
