LOCUS       sequence-226235-        6862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the bacterial expression of the BRET semi-synthetic
            biosensor measuring the NADPH concentration with a C50 of 6.5 uM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6862)
  AUTHORS   Yu Q, Xue L, Hiblot J, Griss R, Fabritz S, Roux C, Binz PA, Haas D, 
            Okun JG, Johnsson K
  TITLE     Semisynthetic sensor proteins enable metabolic assays at the point 
            of care.
  JOURNAL   Science. 2018 Sep 14;361(6407):1122-1126. doi: 
            10.1126/science.aat7992.
  PUBMED    30213915
REFERENCE   2  (bases 1 to 6862)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2018-09-14- 14"; volume: "361"; 
            issue: "6407"; pages: "1122-1126"
COMMENT     Sequence Label: pET51b(+)-NADPH-LUCID-4 (C50 6.5 uM)
FEATURES             Location/Qualifiers
     source          1..6862
                     /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     213..232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        213..231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    232..256
                     /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             271..293
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             309..332
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             339..353
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2070..2099
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(2122..2140)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2136..2183
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2220..2675
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2307..2326)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2517..2538
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2790..3581,3582..3650))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3413..3432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3651..3743)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3824..4412
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4313..4332
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4566..4583
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4598..4737
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4663..4682)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4823..4845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4839..5030)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5602..6684)
                     /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     6646..6665
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6685..6762)
                     /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 atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      241 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
      301 tggcaagctg gagccacccg cagttcgaaa agggtgcaga tgacgacgac aaggtaccgc
      361 atatggataa agactgcgaa atgaaacgca ctaccctgga cagcccgctg ggtaaactgg
      421 aactgagcgg ttgcgagcag ggcctgcacg aaatcatttt cctgggcaaa ggtacgagcg
      481 ctgcggatgc ggtcgaagta ccggctccgg ctgcggttct gggtggcccg gagccgctga
      541 tgcaggctac cgcatggctg aacgcatatt ttcaccagcc agaggcgatt gaagagttcc
      601 ctgtaccggc cctgcatcac ccggtatttc agcaggaatc ctttacccgt caggtactgt
      661 ggaaactgct gaaagtagtg aaattcggcg aggtgatctc ctattcccat ctggcggcgc
      721 tggctggcaa cccagcggca actgcggcgg tgaaaaccgc tctgtctggt aatccggtgc
      781 caattctgat cccgtgccac cgtgtggttc agggcgacct ggacgtaggt ggctacgaag
      841 gcggcctggc tgtgaaagaa tggctgctgg cgcacgaagg tcaccgtctg ggtaaaccag
      901 gcctgggcga attccctcct cctccgccac cgccaccacc gccgccgccg ccaccaccac
      961 ctcctccgcc tcctccacca ccaccgccac cgcctccacc acctggcggc cgcatgatca
     1021 gtctgattgc ggcgttagcg gtagatcgcg ttatcggcat ggaaaacgcc atgccgtgga
     1081 acggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg
     1141 tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta
     1201 cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca
     1261 aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga
     1321 tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc
     1381 tgtgcgaacg cattctggcg ggtggcaccg gtggctctgg cggtaccggt ggctctatgg
     1441 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
     1501 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
     1561 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
     1621 tcccgtatga actgcctgcc gatctcgcct ggtttaaacg caacacctta aataaacccg
     1681 tgattatggg cgcacaaacc tgggaatcaa tcggtcgtcc gttgccagga cgcaaaaata
     1741 ttatcctcag cagtcaaccg ggtacggacg atcgcgtaac gtgggtgaag tcggtggatg
     1801 aagccatcgc ggcgtgtggt gacgtaccag aaatcatggt gattggcggc ggtcgcgttt
     1861 atgaacagtt cttgccaaaa gcgcaaaaac tgtatctgac gcatatcgac gcagaagtgg
     1921 aaggcgacac ccatttcccg gattacgagc cggatgactg ggaatcggta ttcagcgaat
     1981 tccacgatgc tgatgcgcag aactctcaca gctattgctt tgagattctg gagcggcggg
     2041 gagctccggg cttctcctca atttccgctc atcaccacca tcatcaccat caccaccact
     2101 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2161 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2221 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2281 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2341 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2401 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2461 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2521 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2581 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2641 cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta
     2701 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2761 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2821 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2881 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2941 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3001 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3061 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3121 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3181 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3241 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3301 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3361 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3421 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3481 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3541 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3601 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3661 tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3721 atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt
     3781 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3841 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3901 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3961 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4021 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4081 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4141 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4201 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4261 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4321 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4381 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4441 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4501 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4561 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4621 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4681 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4741 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4801 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4861 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4921 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4981 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     5041 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     5101 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     5161 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     5221 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     5281 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5341 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5401 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5461 gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg
     5521 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5581 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5641 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5701 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5761 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5821 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5881 gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5941 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     6001 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     6061 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     6121 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     6181 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     6241 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     6301 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6361 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     6421 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     6481 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6541 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6601 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6661 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6721 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6781 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6841 ccgccgcaag gaatggtgca tg
//
