LOCUS       sequence-153325-        5574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Pl-B biosensor under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5574)
  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 5574)
  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-Pl-B
FEATURES             Location/Qualifiers
     source          1..5574
                     /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(514..533)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(515..533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(617..636)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        842..919
                     /gene="lacI"
                     /label=lacI promoter
     CDS             920..2002
                     /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(939..958)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2811..3002
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2996..3018)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3104..3246
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3159..3178
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3261..3278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3512..3531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4185..4976,4977..5045))
                     /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     4808..4827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5046..5150)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5176..5574,1..57))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5313..5334)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5525..5544
                     /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 ggtagccagg ctgccattga cggttcatca agaaggccct gtggctttgc gtccttatct
      421 ttcgacaagt ttgccgttat cgaagtggac ccgtccttca ccatttcatt cagttacatc
      481 cggccgctgc tctaccgact gagctatccg ggcccctata gtgagtcgta ttaatttcgc
      541 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      601 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      661 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      721 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
      781 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
      841 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      901 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
      961 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1021 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1081 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1141 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1201 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1261 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1321 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1381 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1441 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1501 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1561 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1621 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1681 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     1741 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1801 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1861 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1921 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1981 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2041 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2101 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2161 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2221 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2281 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2341 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     2401 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     2461 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     2521 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     2581 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     2641 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     2701 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     2761 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     2821 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     2881 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     2941 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3001 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3061 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3121 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3181 gagagtgcac catatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3241 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3301 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3361 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3421 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3481 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3541 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3601 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3661 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3721 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3781 gctggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3841 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     3901 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3961 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4021 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4081 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4141 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4201 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4261 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4321 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4381 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4441 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4501 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4561 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4621 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4681 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4741 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4801 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4861 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4921 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4981 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5041 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5101 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5161 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5221 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5281 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5341 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5401 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5461 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5521 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcgg
//
