LOCUS       sequence-149374-        4709 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses inactive WspR G249A under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4709)
  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 4709)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pCOLA-T7-WspR:G249A
FEATURES             Location/Qualifiers
     source          1..4709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             1356..1400
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1438..1456)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1452..1499
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1729..2544)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1729..2544)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2452..2471
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2545..2636)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2654..3289)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2799..2818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3487..4569)
                     /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     4531..4550
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4570..4647)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4693..4709,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgcacaaccc tcatgagagc aagaccgacc tgggcgcccc tctggacggc gcggtcatgg
      361 tactgcttgt cgatgatcag gccatgatcg gagaggctgt gcgtcgctca ctggcgagcg
      421 aggcgggcat cgacttccat ttctgttccg atccgcagca ggcggtggcg gtggccaacc
      481 agatcaagcc gacggtgatc ctccaggacc tggtgatgcc cggcgtcgac ggcctcacgc
      541 tgctcgctgc ctaccgcggc aacccggcga cccgcgacat cccgatcatc gtcctgtcga
      601 ccaaggaaga gccgacggtg aagagcgcgg cgttcgccgc cggcgccaac gactacctgg
      661 tcaagctgcc ggacgccatc gagctggtgg cgcggatccg ctaccactcg cgttcgtaca
      721 tcgccctgca gcaacgcgac gaggcctatc gggcgctgcg cgagagccag cagcagttgc
      781 tggagaccaa cctggtgctg cagcggctga tgaactccga cggcctgacc gggctctcca
      841 accgtcgtca cttcgacgaa tacctggaga tggagtggcg ccgctcgctg cgcgagcagt
      901 cgcaactgtc gctgctgatg atcgacgtgg actatttcaa gagctacaac gacaccttcg
      961 gccacgtcgc cggcgacgag gccctgcgcc aggtcgccgg ggccatccgc gagggctgca
     1021 gtcgctcctc ggacctggcg gctcggtacg gcgccgagga gttcgccatg gtcctcccgg
     1081 gcacctcgcc gggcggcgca cggttgctgg cggagaaggt ccggcgcacg gtggagagcc
     1141 tgcagatcag ccacgaccag ccacgcccgg gctcgcacct gaccgtcagc atcggcgtca
     1201 gcaccctggt gccgggcggc ggcgggcaaa ccttccgggt tctcatcgag atggccgacc
     1261 aggcgctcta ccaggccaag aacaatggcc gcaaccaggt gggcctgatg gaacagccgg
     1321 tgccgccggc cccggcgggc tgactcgagt ctggtaaaga aaccgctgct gcgaaatttg
     1381 aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg
     1441 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1501 tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     1561 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgacaagct
     1621 gacgaccggg tctccgcaag tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1681 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     1741 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1801 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1861 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1921 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1981 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     2041 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2101 acgaaatacg cggtcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     2161 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     2221 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     2281 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     2341 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     2401 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     2461 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     2521 cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca
     2581 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2641 catgctagcg cagaaacgtc ctagaagatg ccaggaggat acttagcaga gagacaataa
     2701 ggccggagcg aagccgtttt tccataggct ccgcccccct gacgaacatc acgaaatctg
     2761 acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2821 tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg
     2881 ctttacccaa atcaccacgt cccgttccgt gtagacagtt cgctccaagc tgggctgtgt
     2941 gcaagaaccc cccgttcagc ccgactgctg cgccttatcc ggtaactatc atcttgagtc
     3001 caacccggaa agacacgaca aaacgccact ggcagcagcc attggtaact gagaattagt
     3061 ggatttagat atcgagagtc ttgaagtggt ggcctaacag aggctacact gaaaggacag
     3121 tatttggtat ctgcgctcca ctaaagccag ttaccaggtt aagcagttcc ccaactgact
     3181 taaccttcga tcaaaccgcc tccccaggcg gttttttcgt ttacagagca ggagattacg
     3241 acgatcgtaa aaggatctca agaagatcct ttacggattc ccgacaccat cactctagat
     3301 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     3361 gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     3421 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     3481 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3541 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3601 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3661 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3721 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     3781 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3841 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3901 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3961 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4021 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4081 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4141 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     4201 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4261 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4321 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     4381 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     4441 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     4501 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4561 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4621 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     4681 gcattaggaa attaatacga ctcactata
//
