LOCUS       sequence-149370-        5598 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses the Dp biosensor under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5598)
  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 5598)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-Dp
FEATURES             Location/Qualifiers
     source          1..5598
                     /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..278)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(537..556)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(538..556)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(640..659)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        865..942
                     /gene="lacI"
                     /label=lacI promoter
     CDS             943..2025
                     /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(962..981)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2834..3025
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3019..3041)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3127..3269
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3182..3201
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3284..3301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3535..3554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4208..4999,5000..5068))
                     /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     4831..4850
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5069..5173)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5200..5598,1..57))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5337..5358)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5549..5568
                     /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 ttatgctatg gcgcccgaac agggacttga
      301 accctggacc cgcggccgga tgtaactagt tacggagctc acactctact caacaacttc
      361 tttcggtggc ccggccaact tccgtgctgc cctgagagaa gagcggagca gcggagttcc
      421 gtgggctttg cgtccccccc tcgcggaggg tttgcctttg tcgagaagtg gacccgtcct
      481 tcaccatttc attcagttac atccggccgc tgctctaccg actgagctat ccgggcccct
      541 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
      601 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
      661 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
      721 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
      781 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
      841 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
      901 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
      961 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     1021 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     1081 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     1141 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     1201 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     1261 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     1321 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     1381 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     1441 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     1501 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     1561 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     1621 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     1681 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     1741 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     1801 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     1861 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     1921 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     1981 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     2041 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     2101 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     2161 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     2221 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     2281 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     2341 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     2401 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     2461 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     2521 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     2581 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     2641 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     2701 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     2761 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     2821 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     2881 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     2941 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     3001 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3061 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3121 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     3181 gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa ataccgcaca
     3241 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     3301 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3361 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3421 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3481 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3541 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3601 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3661 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3721 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3781 gacacgactt atcgccactg gcagctggta acaggattag cagagcgagg tatgtaggcg
     3841 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3901 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3961 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4021 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4081 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4141 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4201 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4261 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4321 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4381 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4441 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4501 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4561 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4621 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4681 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4741 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4801 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4861 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4921 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4981 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5041 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5101 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5161 taggggttcc gcgcacattt ccccgaaaag tgccacctga aattgtaaac gttaatattt
     5221 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5281 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     5341 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5401 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     5461 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5521 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5581 cgctggcaag tgtagcgg
//
