LOCUS       sequence-156461-        5492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Ct biosensor under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5492)
  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 5492)
  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: pET31b-T7-Spinach2-Ct
FEATURES             Location/Qualifiers
     source          1..5492
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(94..141)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     137..155
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(208..225)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      complement(232..279)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(447..465)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(535..554)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        760..837
                     /gene="lacI"
                     /label=lacI promoter
     CDS             838..1920
                     /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"
     primer_bind     complement(857..876)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2729..2920
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2914..2936)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3022..3164
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3077..3096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3179..3196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3430..3449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4103..4894,4895..4963))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4726..4745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4964..5068)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5094..5492,1..57))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5231..5252)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5443..5462
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
       61 attcgccaat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga
      121 ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt
      181 tcgggctttg ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gcaaaaaacc
      241 cctcaagacc cgtttagagg ccccaagggg ttatgctaga tgtaactagt tacggagctc
      301 acactctact caacaaatgg cagcctggcg atcatagctt taaaacattg ttttaaaaca
      361 gtagacccat ggctttgcgt catcaccttt cggtgatttt gccctgtttc attggacccg
      421 tccttcacca tttcattcag ttacatccta tagtgagtcg tattaagatc tcgatcctct
      481 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
      541 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
      601 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
      661 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
      721 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
      781 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
      841 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
      901 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
      961 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1021 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1081 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1141 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1201 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1261 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     1321 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     1381 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     1441 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     1501 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     1561 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     1621 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     1681 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     1741 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     1801 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     1861 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     1921 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     1981 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2041 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     2101 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     2161 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     2221 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     2281 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     2341 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     2401 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     2461 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     2521 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     2581 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     2641 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     2701 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     2761 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     2821 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     2881 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     2941 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3001 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3061 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt
     3121 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3181 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3241 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3301 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3361 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3421 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3481 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3541 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3601 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3661 agtccaaccc ggtaagacac gacttatcgc cactggcagc tggtaacagg attagcagag
     3721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3781 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4381 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     4441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     5101 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     5161 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     5221 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     5281 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     5341 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5401 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5461 agcgggcgct agggcgctgg caagtgtagc gg
//
