LOCUS       sequence-308579-        7202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid used to express the tVYN-Nt∆ luminescent biosensor for use 
            in live cell cyclic di-GMP detection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  AUTHORS   Dippel AB, Anderson WA, Park JH, Yildiz FH, Hammond MC
  TITLE     Development of Ratiometric Bioluminescent Sensors for in Vivo 
            Detection of Bacterial Signaling.
  JOURNAL   ACS Chem Biol. 2020 Mar 25. doi: 10.1021/acschembio.9b00800.
  PUBMED    32186367
REFERENCE   2  (bases 1 to 7202)
  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: "ACS Chem 
            Biol. 2020 Mar 25. doi: 10.1021/acschembio.9b00800."
COMMENT     Sequence Label: pET21-tVYN-Nt∆
FEATURES             Location/Qualifiers
     source          1..7202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(297..299,300..302,303..815)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(342..363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(603..622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     950..971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2142..2159
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2212..2230)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2226..2273
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2310..2765
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2397..2416)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2607..2628
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2791..2895
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2896..2964,2965..3756)
                     /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     complement(3114..3133)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3927..4515
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4416..4435
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4669..4686
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4701..4840
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4766..4785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4926..4948
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4942..5133)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5942..7024)
                     /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     6986..7005
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7025..7102)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      361 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
      421 agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      481 tgaccaccct gggctacggc ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc
      541 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
      601 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      661 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
      721 tggagtacaa ctacaacagc cacaacgtct atatcaccgc cgacaagcag aagaacggca
      781 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc
      841 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      901 tgagctacca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
      961 tggagttcgt gaccgccggt acaggtagca ttaaaattcg cgtggataac aaagattata
     1021 gcagccgcat tgaagatatg gatagcgatt atctgtatat tagcaccccg atggaaaaag
     1081 gccagctggt gcattttagc cagggcagca aaattagcgt gtatattatt gtgaaaggcg
     1141 cggtgtataa ctttgaagaa aaaattaaag aacagattaa aagcccggtg ccgctgctga
     1201 aaattagcaa accggataaa ctgaaaaaaa ttcagcgccg ccagtttttt cgcctggaaa
     1261 aaaaactgcc ggtgaaatat aaaattctgg atgatgattg cgaaagcgaa ctgagcgata
     1321 ccaaagatgc gtatgcgctg gatattagcg gcggcggcct gaaactggcg acccaggaaa
     1381 ttattccggt gaacagcttt ctggaactga actttgaact gaacattgat gaaggcaaaa
     1441 acagcaacat tcatgatatt cgctgcgtgg gcaaaattgt gcgcacccag aaagtggata
     1501 ccgatcgcgt gagcatttat cattatggcg tgaaatttat tagcctgccg agcgaaattc
     1561 aggataccat tgtgcgcttt atttttaacg aacagcgcaa actgcgcctg ggtggtaccc
     1621 tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg
     1681 aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa
     1741 ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg
     1801 aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg
     1861 tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta
     1921 cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca
     1981 aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga
     2041 tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc
     2101 tgtgcgaacg cattctggcg aagcttgcgg ccgcactcga gcaccaccac caccaccact
     2161 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     2221 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2281 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2341 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2401 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2461 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2521 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2581 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2641 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2701 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2761 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2821 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2881 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2941 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3001 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3061 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3121 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     3181 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3241 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3301 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3361 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3421 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3481 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3541 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3601 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3661 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3721 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3781 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3841 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3901 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3961 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4021 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4081 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4141 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4201 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4261 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4321 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4381 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4441 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4501 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4561 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4621 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4681 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4741 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4801 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4861 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4921 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4981 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5041 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5101 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5161 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5221 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5281 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5341 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5401 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5461 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5521 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5581 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5641 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5701 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5761 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5821 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5881 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5941 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6001 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6061 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6121 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6181 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     6241 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6301 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6361 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6421 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6481 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6541 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6601 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6661 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6721 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6781 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6841 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6901 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6961 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     7021 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7081 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     7141 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     7201 tg
//
