LOCUS       sequence-243988-        6357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For TEV-based leakiness regulation. pSB4A3 carrying 
            ParsR-ABS62-32ECF11-t-Pecf11-32malE-te-TEV-t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6357)
  AUTHORS   Wan X, Volpetti F, Petrova E, French C, Maerkl SJ, Wang B
  TITLE     Cascaded amplifying circuits enable ultrasensitive cellular sensors 
            for toxic metals.
  JOURNAL   Nat Chem Biol. 2019 May;15(5):540-548. doi: 
            10.1038/s41589-019-0244-3. Epub 2019 Mar 25.
  PUBMED    30911179
REFERENCE   2  (bases 1 to 6357)
  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: 
            "10.1038/s41589-019-0244-3"; journalName: "Nat Chem Biol"; date: 
            "2019-05"; volume: "15"; issue: "5"; pages: "540-548"
COMMENT     Sequence Label: PXWABS62(E11-TEV)
FEATURES             Location/Qualifiers
     source          1..6357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..59
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(362..1312)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1360..1582
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(2074..2168)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(2198..2989,2990..3058))
                     /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     2821..2840
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3059..3163)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3228..3246
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3250..3293)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3296..3317
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      4092..4163
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4179..4206
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             4308..5405
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      5262..5271
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5379..5402
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5463..5483
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5487..6194
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      6217..6288
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6304..6331
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    join(6338..6357,1)
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
ORIGIN
        1 gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat
       61 tacttcgcgt tatgcaggct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatctcgag gttacattgt cgatctgttc
      181 atggtgaaca gctttgaatg caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt
      241 tacaccgttt tcatctgtgc atatggacag ttttcccttt gatatgtaac ggtgaacagt
      301 tgttctactt ttgtttgtta gtcttgatgc ttcactgata gatacaagag ccataagaac
      361 ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt ttttgcgtga
      421 gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa aattttgcct
      481 caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt cttagtccgt
      541 tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc atttttatct
      601 ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact tggaaaatca
      661 acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg taagtgttta
      721 aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca tggtagttat
      781 tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt gccttgtgag
      841 ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag tatttgtttt
      901 caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg aaaagataag
      961 gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg gcatagtttg
     1021 tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca gttctcgtca
     1081 tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg atgttcatca
     1141 tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta gggttttcaa
     1201 tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc tccgttaagt
     1261 catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac atacatctca
     1321 attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa tgataattac
     1381 atgtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt gctggaaaac
     1441 ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt ttttttgttt
     1501 atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata aaaagaatag
     1561 atcccagccc tgtgtataac tcactacttt agtcagttcc gcagtattac aaaaggatgt
     1621 cgcaaacgct gtttgctcct ctacaaaaca gaccttaaaa ccctaaaggc ttaagtagca
     1681 ccctcgcaag ctcgggcaaa tcgctgaata ttccttttgt ctccgaccat caggcacctg
     1741 agtcgctgtc tttttcgtga cattcagttc gctgcgctca cggctctggc agtgaatggg
     1801 ggtaaatggc actacaggcg ccttttatgg attcatgcaa ggaaactacc cataatacaa
     1861 gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg gtctgctatg tggtgctatc
     1921 tgactttttg ctgttcagca gttcctgccc tctgattttc cagtctgacc acttcggatt
     1981 atcccgtgac aggtcattca gactggctaa tgcacccagt aaggcagcgg tatcatcaac
     2041 aggcttaccc gtcttactgt ccctagtgct tggattctca ccaataaaaa acgcccggcg
     2101 gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa
     2161 caggagtcca agcgagctcg taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2221 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2281 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2341 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2401 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2461 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2521 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2581 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2641 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2701 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2761 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2821 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2881 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     2941 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3001 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3061 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3121 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt cccatggtgc
     3181 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3241 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggaatt
     3301 cgcggccgct tctagagcca actcaaaatt cacacctatt accttcctct gcacttacac
     3361 attcgttaag tcatatatgt ttttgactta tccgcttcga agagagacac tacctgcaaa
     3421 cttacacata agtcatatat gtttttgact tatccgcttc tactagagtc acacaggaaa
     3481 gtactagatg atgagcgata gtccgcagaa actgggtcgt aatgaatgga atgcatatat
     3541 ggataaagtg aaagccaaag atcgtgaagc ctttgccttt gtgtttcgtt tttatgcacc
     3601 gaaactgaaa cagttcgcct ataaacatgt tggcaatgaa caggttgcaa tggaaatggt
     3661 tcaagaaacc atggcaaccg tttggcagaa agcacatctg tatgatggta aaaaaagcgc
     3721 actgagcacc tggatttata ccattattcg taacctgtgc tttgacctgc tgcgtaaaca
     3781 gaaaggtaaa gaactgcata ttcacagcga cgatatttgg ccgagcgaat attatccgcc
     3841 tgatatggtt gatcattata gtccggaaca ggatatgctg aaagaacagg tggttaaatt
     3901 tctggatatc ctgccgaaaa atcagcgtga tgttctgcaa gcagtttatc tggaagaact
     3961 gccgcatcag caggttgcag aactgtttga tattccgctg ggcaccgtta aaagccgtct
     4021 gcgtctggca gttgaaaaac tgcgtcatag catgcatacc gaacagctgt aataatacta
     4081 gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     4141 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     4201 tctgcgttta tatactagag gcctccacac cgctcgtcac atcctgtgat ccactcttca
     4261 tcccgctacg taacacctct tactagagtc acacaggaaa gtactagatg atcgaagaag
     4321 gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta
     4381 agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag
     4441 agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg
     4501 accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt
     4561 tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg
     4621 cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc
     4681 cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga
     4741 gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg
     4801 ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata
     4861 acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga
     4921 atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga
     4981 ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa
     5041 cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag
     5101 gtattaacgc cgccagtccg aacaaagagc tggcgaaaga gttcctcgaa aactatctgc
     5161 tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga
     5221 agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccaccatg gaaaacgccc
     5281 agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta
     5341 ctgcggtgat caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc
     5401 agactcgtat caccaagggc ggctccggcg gctccggcgg caccggcggc tccggcggct
     5461 ccgaaaacct gtattttcag ggtggcggcg aaagcctgtt caaaggtccg cgtgactaca
     5521 acccgattag ctcgacgatc tgccacctga cgaacgaaag cgacggccac accacgagcc
     5581 tgtatggcat cggttttggc ccgttcatta tcacgaacaa acacctgttt cgtcgcaaca
     5641 atggtaccct gctggtgcag tctctgcatg gcgtgtttaa agttaaaaat accacgaccc
     5701 tgcaacaaca cctgatcgat ggtcgtgaca tgattatcat tcgcatgccg aaagattttc
     5761 cgccgttccc gcagaaactg aaattccgtg aaccgcaacg tgaagaacgc atttgcctgg
     5821 tcacgaccaa ctttcagacc aaatcaatga gctctatggt tagcgatacg tcttgtacct
     5881 ttccgagttc cgacggcatc ttctggaaac attggattca gaccaaagat ggtcaatgcg
     5941 gcagtccgct ggtttccacc cgtgacggtt tcatcgtcgg cattcactca gcgtcgaact
     6001 ttacgaatac caacaattac ttcacgtccg ttccgaaaaa ctttatggaa ctgctgacca
     6061 atcaggaagc gcagcaatgg gtgtcaggtt ggcgcctgaa tgccgattcg gttctgtggg
     6121 gcggtcacaa agtctttatg gtgaaaccgg aagaaccgtt ccagccggtc aaagaagcaa
     6181 cccaactgat gaattaataa tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg
     6241 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     6301 actggctcac cttcgggtgg gcctttctgc gtttatatac tagtagcggc cgctgca
//
