LOCUS       sequence-297926-        5566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SmtB protein under the control of T7-lacO promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5566)
  AUTHORS   Jung JK, Alam KK, Verosloff MS, Capdevila DA, Desmau M, Clauer PR, 
            Lee JW, Nguyen PQ, Pasten PA, Matiasek SJ, Gaillard JF, Giedroc DP, 
            Collins JJ, Lucks JB
  TITLE     Cell-free biosensors for rapid detection of water contaminants.
  JOURNAL   Nat Biotechnol. 2020 Jul 6. pii: 10.1038/s41587-020-0571-7. doi: 
            10.1038/s41587-020-0571-7.
  PUBMED    32632301
REFERENCE   2  (bases 1 to 5566)
  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: "Nat 
            Biotechnol. 2020 Jul 6. pii: 10.1038/s41587-020-0571-7. doi: 
            10.1038/s41587-020-0571-7."
COMMENT     Sequence Label: pJBL725
FEATURES             Location/Qualifiers
     source          1..5566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             435..455
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             456..473
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(482..500)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      496..543
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      580..1035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(667..686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     877..898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1127..1942)
                     /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(1127..1942)
                     /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     1850..1869
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1934..1953
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2064..2652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2553..2572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2806..2823
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2838..2977
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2903..2922)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3063..3085
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3079..3270)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4079..5161)
                     /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     5123..5142
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5162..5239)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5445..5464
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5548..5566,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5548..5566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gaccaagcct gtattacaag atggtgaaac ggtcgtatgt caaggaacac
      121 atgctgctat cgcctcagaa ttgcaagcga ttgctcccga ggttgcgcag tccctggcag
      181 agttttttgc agtccttgct gatcccaacc gtttacgttt attgtcgtta ttggcacgtt
      241 ctgagctgtg cgtcggcgac ttagcgcagg caatcggtgt gagcgaatct gcggttagtc
      301 atcagttgcg tagtttgcgc aatctgcgct tggtctcgta tcgtaaacag gggcgtcatg
      361 tgtattacca acttcaagat caccatatcg ttgccttata tcagaatgct ttggaccatc
      421 tgcaagaatg tcgtgagaat ctttattttc aagggcatca ccatcatcat cattaactcg
      481 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      541 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
      601 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      661 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      721 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      781 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
      841 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
      901 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
      961 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1021 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1081 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1141 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1201 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1261 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1321 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1381 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1441 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1501 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1561 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     1621 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     1681 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     1741 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     1801 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     1861 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     1921 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     1981 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2041 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2101 caaacaaaaa aaccaccact accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2161 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2221 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2281 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2341 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2401 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2461 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2521 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2581 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2641 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2701 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2761 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2821 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2881 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     2941 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3001 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3061 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3121 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3181 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3241 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3301 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3361 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3421 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3481 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3541 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     3601 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     3661 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     3721 gatcatgcgc acccatgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     3781 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     3841 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     3901 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     3961 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4021 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4081 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4141 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4201 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4261 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4321 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     4381 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4441 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4501 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4561 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4621 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     4681 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     4741 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     4801 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     4861 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     4921 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     4981 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5041 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5101 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5161 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5221 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5281 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5341 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5401 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5461 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5521 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagg
//
