LOCUS       sequence-153952-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression of Ct biosensor and YhjH under individual T7 
            promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  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 6278)
  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-YhjH
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     623..642
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(662..739)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     945..964
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1052..1070
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1071..1095
                     /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             1110..1132
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        1972..1990
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      2234..2280
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             2292..2336
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2374..2392)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2388..2435
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2472..2927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2559..2578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2769..2790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3042..3833,3834..3902))
                     /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     3665..3684
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3903..3995)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4076..4664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4565..4584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4818..4835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4850..4992
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4918..4937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5078..5100
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5094..5285)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5857..6278,1..661))
                     /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 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
       61 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
      121 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      181 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      241 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      301 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      361 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      421 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      481 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      541 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      601 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      661 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      721 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      781 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      841 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      901 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      961 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     1021 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
     1081 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatataccta
     1141 tgataaggca ggttatccag cgaataagca accctgaagc aagcatcgag agcttgcagg
     1201 aacggcgttt ttggttgcag tgtgagcgtg cttacacctg gcagccgatc tatcaaacat
     1261 gcgggcggtt aatggccgtg gagctattaa cggtggtcac gcatcccttg aacccttcgc
     1321 aacgcctgcc gccggatcgc tattttactg aaatcaccgt cagccatcgg atggaggttg
     1381 tgaaagagca gattgatttg ctggcgcaaa aagccgactt ctttatagag cacggcctgc
     1441 tggcatcggt caatattgat ggccctacgc tcatcgccct gcgtcagcaa ccaaaaatcc
     1501 tgcgccagat tgagcgtctt ccctggctgc gtttcgaact ggtggagcat atccgtctgc
     1561 cgaaagattc aacctttgcc tcgatgtgtg aatttggccc gctgtggctg gatgattttg
     1621 gtaccgggat ggcaaatttc tctgcgctaa gtgaagtgcg ttatgactac atcaaaatcg
     1681 cgcgagaact gtttgtgatg ctgcgtcagt cgccggaagg acgcacactc ttttctcagc
     1741 ttttacatct aatgaatcgc tattgtcgcg gggtgattgt cgagggcgta gaaacgccgg
     1801 aagagtggcg tgatgttcag aactcgcccg cattcgccgc acaaggctgg tttctttcac
     1861 gcccggcacc gatagaaacg ctgaatacgg cggttctggc gctataatgc ttaagtcgaa
     1921 cagaaagtcg tattgtacac ggccgcataa tcgaaatcga tcccgcgaaa ttaatacgac
     1981 tcactatagg ggcccggata gctcagtcgg tagagcagcg gccggatgta actgaatgaa
     2041 atggtgaagg acgggtccaa tgaaacaggg caaaatcacc gaaaggtgat gacgcaaagc
     2101 catgggtcta ctgttttaaa acaatgtttt aaagctatga tcgccaggct gccatttgtt
     2161 gagtagagtg tgagctccgt aactagttac atccggccgc gggtccaggg ttcaagtccc
     2221 tgttcgggcg ccatagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2281 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     2341 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     2401 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     2461 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2521 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2581 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2641 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2701 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2761 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2821 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2881 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
     2941 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3001 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3061 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3121 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3181 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3241 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3301 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3361 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3421 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3481 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3541 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3601 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3661 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3721 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3781 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3841 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3901 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     3961 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     4021 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4081 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4141 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4201 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4261 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4321 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4381 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4441 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4501 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4561 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4621 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4681 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4741 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4801 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4861 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     4921 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4981 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5041 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5101 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5161 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5221 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5281 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5341 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5401 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     5461 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     5521 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     5581 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     5641 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     5701 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     5761 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5821 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5881 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5941 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     6001 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6061 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     6121 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6181 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6241 cagcatttgc atggtttgtt gaaaaccgga catggcac
//
