LOCUS       sequence-211907-        6680 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 6680)
  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 6680)
  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: pCdrA-gfp(ASV)C
FEATURES             Location/Qualifiers
     source          1..6680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..50)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(204..792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(284..303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(963..1754,1755..1823))
                     /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     1586..1605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1824..1928)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1996..2014
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2052..2074)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2174..2193
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2285..2818)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2988..2993,2994..3010,3011..3016))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      3066..3417
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             3431..4264
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     primer_bind     4410..4432
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4424..4441
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4425..4441
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             4694..4705
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4718..5431
                     /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             4718..5431
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4748..4776)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5371..5391
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5496..5590
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             5634..6293
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5700..5719)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6358..6444
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(6482..6498)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6482..6498)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6495..6517)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6506..6522
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6530..6536,6537..6554,6555..6560))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6575..6596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
       61 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
      121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
      781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
      841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
      901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
      961 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     1021 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     1081 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     1141 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     1201 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     1261 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     1321 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     1381 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     1441 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     1501 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     1561 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     1621 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     1681 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     1741 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     1801 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1861 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1921 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     1981 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     2041 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     2101 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2161 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatcg aattggccgc
     2221 ggcgttgtga caatttaccg aacaactccg cggccgggaa gccgatctcg gcttgaacga
     2281 attgttaggt ggcggtactt gggtcgatat caaagtgcat cacttcttcc cgtatgccca
     2341 actttgtata gagagccact gcgggatcgt caccgtaatc tgcttgcacg tagatcacat
     2401 aagcaccaag cgcgttggcc tcatgcttga ggagattgat gagcgcggtg gcaatgccct
     2461 gcctccggtg ctcgccggag actgcgagat catagatata gatctcacta cgcggctgct
     2521 caaacttggg cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg tcgaaggcag
     2581 caagcgcgat gaatgtctta ctacggagca agttcccgag gtaatcggag tccggctgat
     2641 gttgggagta ggtggctacg tctccgaact cacgaccgaa aagatcaaga gcagcccgca
     2701 tggatttgac ttggtcaggg ccgagcctac atgtgcgaat gatgcccata cttgagccac
     2761 ctaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctc cataacatca
     2821 aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtacaa
     2881 aaaaacagtc ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg ctgcgttcgg
     2941 tcaaggttct ggaccagttg cgtgagcgca tacgctactt gcattacagt ttacgaaccg
     3001 aacaggctta tgtcaattcg cctctcaggc gccgctggtg ccgctggttg gacgccaagg
     3061 gtgaatccgc ctcgataccc tgattactcg cttcctgcgc cctctcaggc ggcgataggg
     3121 gactggtaaa acggggattg cccagacgcc tcccccgccc cttcaggggc acaaatgcgg
     3181 ccccaacggg gccacgtagt ggtgcgtttt ttgcgtttcc acccttttct tccttttccc
     3241 ttttaaacct tttaggacgt ctacaggcca cgtaatccgt ggcctgtaga gtttaaaaag
     3301 ggacggattt gttgccatta agggacggat ttgttgttaa gaagggacgg atttgttgtt
     3361 gtaaagggac ggatttgttg tattgtggga cgcagataca gtgtcccctt atacacaagg
     3421 aatgtcgaac gtggcctcac ccccaatggt ttacaaaagc aatgccctgg tcgaggccgc
     3481 gtatcgcctc agtgttcagg aacagcggat cgttctggcc tgtattagcc aggtgaagag
     3541 gagcgagcct gtcaccgatg aagtgatgta ttcagtgacg gcggaggaca tagcgacgat
     3601 ggcgggtgtc cctatcgaat cttcctacaa ccagctcaaa gaagcggccc tgcgcctgaa
     3661 acggcgggaa gtccggttaa cccaagagcc caatggcaag gggaaaagac cgagtgtgat
     3721 gattaccggc tgggtgcaaa caatcatcta ccgggagggt gagggccgtg tagaactcag
     3781 gttcaccaaa gacatgctgc cgtacctgac ggaactcacc aaacagttca ccaaatacgc
     3841 cttggctgac gtggccaaga tggacagcac ccacgcgatc aggctttacg agctgctcat
     3901 gcaatgggac agcatcggcc agcgcgaaat agaaattgac cagctgcgaa agtggtttca
     3961 actggaaggc cggtatccct cgatcaagga cttcaagttg cgagtgcttg atccagccgt
     4021 gacgcagatc aacgagcaca gcccgctaca ggtggagtgg gcgcagcgaa agaccgggcg
     4081 caaggtcaca catctgttgt tcagttttgg accgaagaag cccgccaagg cggtgggtaa
     4141 ggccccagcg aagcgcaagg ccgggaagat ttcagatgct gagatcgcga aacaggctcg
     4201 ccctggtgag acatgggaag cggcccgcgc tcgactaacc cagatgccgc tggatctggc
     4261 ctagaggccg tggccaccac ggcccggcct gcctttcagg ctgcgcaact gttgggaagg
     4321 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     4381 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4441 tgccaagctg ggcggccgcg cgaattcgag ctcggtaccc ggggatcctc tagacattgt
     4501 cggttttttg acggtattac atatttaacc tccgtagtgt catttttgag tcactccgac
     4561 gaacggtcgc cctttcaagt ggacaatttt tgatctgaaa ggcatttttt tggcacacaa
     4621 ggcgcaatgt gaattgcttc tcagattttc gtcatttaac tgacgaaatg cagtgctgga
     4681 aaactgacgt gcgaaagagg agaaattaac tatgagcatg cgtaaaggag aagaactttt
     4741 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
     4801 tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg
     4861 cactactgga aaactacctg ttccatggcc aacacttgtc actactttcg gttatggtgt
     4921 tcaatgcttt gcgagatacc cagatcatat gaaacagcat gactttttca agagtgccat
     4981 gcccgaaggt tatgtacagg aaagaactat atttttcaaa gatgacggga actacaagac
     5041 acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat
     5101 tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact ataactcaca
     5161 caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagttaact tcaaaattag
     5221 acacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccaat
     5281 tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc
     5341 gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg
     5401 gattacacat ggcatggatg aactatacaa aaggcctgca gcaaacgacg aaaactacgc
     5461 tgcatcagtt taataagctt aattagctga gcttggactc ctgttgatag atccagtaat
     5521 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     5581 tggtgagaat ccaagctagc ttggcgagat tttcaggagc taaggaagct aaaatggaga
     5641 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     5701 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     5761 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     5821 ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac ggtgagctgg
     5881 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     5941 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     6001 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     6061 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     6121 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     6181 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     6241 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt
     6301 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa
     6361 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     6421 aacgctctcc tgagtaggac aaatccgccg ctaggagctt gcggccgcaa ttcgtaatca
     6481 tgtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     6541 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     6601 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6661 tcggccaacg cgcggggaga
//
