LOCUS       sequence-211903-        6638 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 6638)
  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 6638)
  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-gfpC
FEATURES             Location/Qualifiers
     source          1..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(35..568)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(738..743,744..760,761..766))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      816..1167
                     /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             1181..2014
                     /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     2160..2182
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2174..2191
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2175..2191
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             2444..2455
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2468..3181
                     /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(2498..2526)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3121..3141
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3204..3298
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3342..4001
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3408..3427)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4066..4152
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(4190..4206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4190..4206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4203..4225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4214..4230
                     /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(4238..4244,4245..4262,4263..4268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4283..4304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4421..4438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4592..5180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4672..4691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5351..6142,6143..6211))
                     /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     5974..5993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6212..6316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6384..6402
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6440..6462)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6562..6581
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt gggtcgatat
       61 caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact gcgggatcgt
      121 caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc tcatgcttga
      181 ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctcgccggag actgcgagat
      241 catagatata gatctcacta cgcggctgct caaacttggg cagaacgtaa gccgcgagag
      301 cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta ctacggagca
      361 agttcccgag gtaatcggag tccggctgat gttgggagta ggtggctacg tctccgaact
      421 cacgaccgaa aagatcaaga gcagcccgca tggatttgac ttggtcaggg ccgagcctac
      481 atgtgcgaat gatgcccata cttgagccac ctaactttgt tttagggcga ctgccctgct
      541 gcgtaacatc gttgctgctc cataacatca aacatcgacc cacggcgtaa cgcgcttgct
      601 gcttggatgc ccgaggcata gactgtacaa aaaaacagtc ataacaagcc atgaaaaccg
      661 ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca
      721 tacgctactt gcattacagt ttacgaaccg aacaggctta tgtcaattcg cctctcaggc
      781 gccgctggtg ccgctggttg gacgccaagg gtgaatccgc ctcgataccc tgattactcg
      841 cttcctgcgc cctctcaggc ggcgataggg gactggtaaa acggggattg cccagacgcc
      901 tcccccgccc cttcaggggc acaaatgcgg ccccaacggg gccacgtagt ggtgcgtttt
      961 ttgcgtttcc acccttttct tccttttccc ttttaaacct tttaggacgt ctacaggcca
     1021 cgtaatccgt ggcctgtaga gtttaaaaag ggacggattt gttgccatta agggacggat
     1081 ttgttgttaa gaagggacgg atttgttgtt gtaaagggac ggatttgttg tattgtggga
     1141 cgcagataca gtgtcccctt atacacaagg aatgtcgaac gtggcctcac ccccaatggt
     1201 ttacaaaagc aatgccctgg tcgaggccgc gtatcgcctc agtgttcagg aacagcggat
     1261 cgttctggcc tgtattagcc aggtgaagag gagcgagcct gtcaccgatg aagtgatgta
     1321 ttcagtgacg gcggaggaca tagcgacgat ggcgggtgtc cctatcgaat cttcctacaa
     1381 ccagctcaaa gaagcggccc tgcgcctgaa acggcgggaa gtccggttaa cccaagagcc
     1441 caatggcaag gggaaaagac cgagtgtgat gattaccggc tgggtgcaaa caatcatcta
     1501 ccgggagggt gagggccgtg tagaactcag gttcaccaaa gacatgctgc cgtacctgac
     1561 ggaactcacc aaacagttca ccaaatacgc cttggctgac gtggccaaga tggacagcac
     1621 ccacgcgatc aggctttacg agctgctcat gcaatgggac agcatcggcc agcgcgaaat
     1681 agaaattgac cagctgcgaa agtggtttca actggaaggc cggtatccct cgatcaagga
     1741 cttcaagttg cgagtgcttg atccagccgt gacgcagatc aacgagcaca gcccgctaca
     1801 ggtggagtgg gcgcagcgaa agaccgggcg caaggtcaca catctgttgt tcagttttgg
     1861 accgaagaag cccgccaagg cggtgggtaa ggccccagcg aagcgcaagg ccgggaagat
     1921 ttcagatgct gagatcgcga aacaggctcg ccctggtgag acatgggaag cggcccgcgc
     1981 tcgactaacc cagatgccgc tggatctggc ctagaggccg tggccaccac ggcccggcct
     2041 gcctttcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     2101 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     2161 ccagtcacga cgttgtaaaa cgacggccag tgccaagctg ggcggccgcg cgaattcgag
     2221 ctcggtaccc ggggatcctc tagacattgt cggttttttg acggtattac atatttaacc
     2281 tccgtagtgt catttttgag tcactccgac gaacggtcgc cctttcaagt ggacaatttt
     2341 tgatctgaaa ggcatttttt tggcacacaa ggcgcaatgt gaattgcttc tcagattttc
     2401 gtcatttaac tgacgaaatg cagtgctgga aaactgacgt gcgaaagagg agaaattaac
     2461 tatgagcatg cgtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
     2521 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac
     2581 atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc
     2641 aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc cagatcatat
     2701 gaaacagcat gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat
     2761 atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac
     2821 ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg
     2881 acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa
     2941 gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact
     3001 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
     3061 ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat
     3121 ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa
     3181 ataagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     3241 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     3301 aagctagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     3361 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     3421 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     3481 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     3541 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     3601 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     3661 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     3721 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     3781 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     3841 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     3901 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     3961 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt
     4021 ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat aaaacgaaag
     4081 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4141 agtaggacaa atccgccgct aggagcttgc ggccgcaatt cgtaatcatg tcatagctgt
     4201 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4381 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4441 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4501 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4561 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4621 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4681 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4741 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4801 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4861 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4921 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4981 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5041 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5101 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5161 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5221 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5281 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5341 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5401 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5461 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5521 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5581 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5641 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5701 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5761 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5821 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5881 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5941 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6001 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6061 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6121 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6181 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6241 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6301 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     6361 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     6421 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     6481 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     6541 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatcgaa
     6601 ttggccgcgg cgttgtgaca atttaccgaa caactccg
//
