LOCUS       sequence-149376-        6554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression of Ct biosensor and WspR WT under individual T7 
            promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6554)
  AUTHORS   Wang XC, Wilson SC, Hammond MC
  TITLE     Next-generation RNA-based fluorescent biosensors enable anaerobic 
            detection of cyclic di-GMP.
  JOURNAL   Nucleic Acids Res. 2016 Jul 5. pii: gkw580.
  PUBMED    27382070
REFERENCE   2  (bases 1 to 6554)
  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"; journalName: "Nucleic 
            Acids Res. 2016 Jul 5. pii: gkw580."
COMMENT     Sequence Label: pETDuet-T7-Ct-WspR
FEATURES             Location/Qualifiers
     source          1..6554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     689..708
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(728..805)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1011..1030
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1118..1136
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1137..1161
                     /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)."
     RBS             1176..1198
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        2314..2332
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2576..2622
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2634..2678
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2716..2734)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2730..2777
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2814..3269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2901..2920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3111..3132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3384..4175,4176..4244))
                     /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     4007..4026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4245..4337)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4418..5006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4907..4926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5160..5177
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5192..5334
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5260..5279)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5420..5442
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5436..5627)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6199..6554,1..727))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
       61 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
      121 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
      181 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
      241 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
      301 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
      361 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
      421 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
      481 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
      541 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
      601 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
      661 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
      721 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
      781 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
      841 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
      901 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
      961 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     1021 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     1081 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     1141 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     1201 tacctatgca caaccctcat gagagcaaga ccgacctggg cgcccctctg gacggcgcgg
     1261 tcatggtact gcttgtcgat gatcaggcca tgatcggaga ggctgtgcgt cgctcactgg
     1321 cgagcgaggc gggcatcgac ttccatttct gttccgatcc gcagcaggcg gtggcggtgg
     1381 ccaaccagat caagccgacg gtgatcctcc aggacctggt gatgcccggc gtcgacggcc
     1441 tcacgctgct cgctgcctac cgcggcaacc cggcgacccg cgacatcccg atcatcgtcc
     1501 tgtcgaccaa ggaagagccg acggtgaaga gcgcggcgtt cgccgccggc gccaacgact
     1561 acctggtcaa gctgccggac gccatcgagc tggtggcgcg gatccgctac cactcgcgtt
     1621 cgtacatcgc cctgcagcaa cgcgacgagg cctatcgggc gctgcgcgag agccagcagc
     1681 agttgctgga gaccaacctg gtgctgcagc ggctgatgaa ctccgacggc ctgaccgggc
     1741 tctccaaccg tcgtcacttc gacgaatacc tggagatgga gtggcgccgc tcgctgcgcg
     1801 agcagtcgca actgtcgctg ctgatgatcg acgtggacta tttcaagagc tacaacgaca
     1861 ccttcggcca cgtcgccggc gacgaggccc tgcgccaggt cgccggggcc atccgcgagg
     1921 gctgcagtcg ctcctcggac ctggcggctc ggtacggcgg cgaggagttc gccatggtcc
     1981 tcccgggcac ctcgccgggc ggcgcacggt tgctggcgga gaaggtccgg cgcacggtgg
     2041 agagcctgca gatcagccac gaccagccac gcccgggctc gcacctgacc gtcagcatcg
     2101 gcgtcagcac cctggtgccg ggcggcggcg ggcaaacctt ccgggttctc atcgagatgg
     2161 ccgaccaggc gctctaccag gccaagaaca atggccgcaa ccaggtgggc ctgatggaac
     2221 agccggtgcc gccggccccg gcgggctgat gcttaagtcg aacagaaagt cgtattgtac
     2281 acggccgcat aatcgaaatc gatcccgcga aattaatacg actcactata ggggcccgga
     2341 tagctcagtc ggtagagcag cggccggatg taactgaatg aaatggtgaa ggacgggtcc
     2401 aatgaaacag ggcaaaatca ccgaaaggtg atgacgcaaa gccatgggtc tactgtttta
     2461 aaacaatgtt ttaaagctat gatcgccagg ctgccatttg ttgagtagag tgtgagctcc
     2521 gtaactagtt acatccggcc gcgggtccag ggttcaagtc cctgttcggg cgccatagca
     2581 taaccccttg gggcctctaa acgggtcttg aggggttttt tgtcgagtct ggtaaagaaa
     2641 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     2701 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2761 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2821 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2881 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2941 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3001 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3061 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3121 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3181 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3241 ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag attatcaaaa
     3301 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3361 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3421 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3481 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3541 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3601 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3661 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3721 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3781 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3841 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3901 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3961 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4021 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4081 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4141 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4201 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4261 tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4321 ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt tcgttccact
     4381 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4441 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4501 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4561 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4621 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4681 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4741 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4801 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4861 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4921 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4981 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5041 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5101 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5161 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     5221 tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc
     5281 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5341 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5401 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5461 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5521 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5581 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5641 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5701 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5761 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5821 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5881 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5941 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     6001 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     6061 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     6121 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     6181 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     6241 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     6301 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     6361 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     6421 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6481 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6541 atcgttggca acca
//
