LOCUS       sequence-153317-        5578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Pl-A biosensor under a T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5578)
  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 5578)
  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-A
FEATURES             Location/Qualifiers
     source          1..5578
                     /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(518..537)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(519..537)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(621..640)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        846..923
                     /gene="lacI"
                     /label=lacI promoter
     CDS             924..2006
                     /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(943..962)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2815..3006
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3000..3022)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3108..3250
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3163..3182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3265..3282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3516..3535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4189..4980,4981..5049))
                     /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     4812..4831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5050..5154)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5180..5578,1..57))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5317..5338)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5529..5548
                     /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 gatctcaatg gtggtggtgg tggtgctcaa gcaaaaaacc
      241 cctcaagacc cgtttagagg ccccaagggg ttatgctatg gcgcccgaac agggacttga
      301 accctggacc cgcggccgga tgtaactagt tacggagctc acactctact caacaaccct
      361 tcggtagcca ggctgccatt gacggttcat caagaaggcc ctgtggcttt gcgtccttat
      421 ctttcgacaa gtttgccgtt atcgaagagt ggacccgtcc ttcaccattt cattcagtta
      481 catccggccg ctgctctacc gactgagcta tccgggcccc tatagtgagt cgtattaatt
      541 tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
      601 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
      661 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
      721 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
      781 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
      841 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
      901 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
      961 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     1021 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     1081 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     1141 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     1201 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     1261 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     1321 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     1381 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     1441 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     1501 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     1561 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     1621 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     1681 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     1741 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     1801 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     1861 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     1921 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1981 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     2041 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     2101 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     2161 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     2221 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     2281 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     2341 gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     2401 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     2461 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     2521 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     2581 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     2641 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     2701 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     2761 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     2821 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     2881 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     2941 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     3001 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3061 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     3121 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     3181 tactgagagt gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     3241 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3301 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3361 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3421 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3481 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3541 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3601 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3661 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3721 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3781 ggcagctggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3841 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3901 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3961 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4021 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4081 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4141 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4201 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4261 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4321 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4381 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4441 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4501 tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4561 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     4621 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     4681 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4741 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4801 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4861 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4921 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4981 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5041 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5101 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5161 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5221 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5281 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5341 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5401 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5461 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5521 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcgg
//
