LOCUS       sequence-240600-        6661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For TEV-based leakiness regulation. pSB3K3 carrying 
            J109-30arsR-t-ParsR-30hrpR-30hrpS-t-PhrpLE-30gfp-te-AAV-t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6661)
  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 6661)
  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: pXWJ109As(RS-te-AAV)
FEATURES             Location/Qualifiers
     source          1..6661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(141..956)
                     /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(141..956)
                     /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     864..883
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     948..967
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(1551..2095)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1630..1649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2563..2581
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2585..2628)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2631..2652
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        2653..2687
                     /label=BBa_J23109
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.04"
     RBS             2699..2710
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3133..3204
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3220..3247
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             3375..3386
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4297..4308
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     RBS             4371..4382
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      5317..5388
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5404..5431
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             5665..5676
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5683..6396
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             5683..6396
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(5713..5741)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     6336..6356
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             6442..6462
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      6521..6592
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6608..6635
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    join(6642..6661,1)
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
ORIGIN
        1 ggcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
       61 tcactcaaag gcggtaatct cgagtcccgt caagtcagcg taatgctctg ccagtgttac
      121 aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
      181 ttcatatcag gattatcgat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
      241 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
      301 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
      361 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc
      421 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
      481 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
      541 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
      601 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
      661 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
      721 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
      781 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
      841 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
      901 atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg gctcataaca
      961 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     1021 tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg aataaatcga
     1081 acttttgctg agttgaagga tcagatcacg catcttcccg acaacgcaga ccgttccgtg
     1141 gcaaagcaaa agttcaaaat caccaactgg tccacctaca acaaagctct catcaaccgt
     1201 ggctccctca ctttctggct ggatgatggg gcgattcagg cctggtatga gtcagcaaca
     1261 ccttcttcac gaggcagacc tcagcgctag cggagtgtat actggcttac tatgttggca
     1321 ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg
     1381 tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc
     1441 ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga
     1501 tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg
     1561 ctccgccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     1621 caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc
     1681 ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc
     1741 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
     1801 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
     1861 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
     1921 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
     1981 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcaaaaaac cgccctgcaa
     2041 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
     2101 tcatcttatt aaggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     2221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     2281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     2341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     2401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     2461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattccat ggtgccacct
     2521 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     2581 cagaatttca gataaaaaaa atccttagct ttcgctaagg atgatttctg gaattcgcgg
     2641 ccgcttctag agtttacagc tagctcagtc ctagggactg tgctagctac tagagattaa
     2701 agaggagaaa tactagatgt catttctgtt acccatccaa ttgttcaaaa ttcttgctga
     2761 tgaaacccgt ctgggcatcg ttttactgct cagcgaactg ggagagttat gcgtctgcga
     2821 tctctgcact gctctcgacc agtcgcagcc caagatctcc cgccacctgg cattgctgcg
     2881 tgaaagcggg ctattgctgg accgcaagca aggtaagtgg gttcattacc gcttatcacc
     2941 gcatattcca gcatgggcgg cgaaaattat tgatgaggcc tggcgatgtg aacaggaaaa
     3001 ggttcaggcg attgtccgca acctggctcg acaaaactgt tccggggaca gtaagaacat
     3061 ttgcagttaa aaatttagct aaacacacat gaattttcag atgtgtttta tccgggtact
     3121 agagccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3181 tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct
     3241 ttctgcgttt atatactaga gccaactcaa aattcacacc tattaccttc ctctgcactt
     3301 acacattcgt taagtcatat atgtttttga cttatccgct tcgaagagag acactacctg
     3361 caatactaga gattaaagag gagaaatact agatgagtac aggcatcgat aaggacgtcc
     3421 gagagtgttg gggcgtaact gcattatcag cgggtcatca aattgcaatg aatagcgcgt
     3481 ttctggatat ggacttgctg ttgtgcgggg aaaccggcac cggcaaggac acactggcca
     3541 accgcattca cgagttgtcc agcaggtcgg gaccctttgt gggcatgaac tgcgccgcca
     3601 ttcccgagtc gctggcagag agccagttat tcggtgtggt caacggtgca ttcaccggcg
     3661 tatgccgggc tcgcgagggc tacatagagg cctccagtgg tggcaccttg tacctggatg
     3721 aaatcgacag catgccgttg agcctgcaag ccaaactgct gcgtgtgttg gagagtcgag
     3781 gtatcgagcg tctgggctcg accgaattta tcccggtgga tctgcggatc attgcctcgg
     3841 cccagcggcc actggatgaa ctggtggaac aaggactttt ccgtcgcgac ctgttttttc
     3901 ggctcaacgt gctgacgctt cacttgccag ccttgcgcaa acgtcgtgaa cagatcctgc
     3961 cattgttcga ccagttcacc cagggtatcg ctgccgagtt cggacgtccc gctcctgcgc
     4021 tggacagcgg gcgtgtgcag ctgctgctca gccacgactg gccgggcaac atccgcgaat
     4081 tgaagtctgc ggccaagcgc ttcgtactcg gcttcccctt gctgggcgcc gaccctgtgg
     4141 aagcgcttga ccctgccacg gggctgcgca cgcaaatgcg catcatcgag aaaatgctca
     4201 tccaggatgc cttgaagcgg cacaggcaca atttcgacgc ggtgcttcag gagttggagt
     4261 tgccaagacg caccctgtat caccgcatga aggaactggg agttgcagcg ccgatcgctg
     4321 cgacggccgg ggtctaataa aacgcaggtg tggttatgct actagagatt aaagaggaga
     4381 aatactagat gagtcttgat gaaaggtttg aggatgatct ggacgaggag cgggttccga
     4441 atctggggat agttgccgaa agtatttcgc aactgggtat cgacgtgctg ctatcgggtg
     4501 agaccggcac gggcaaagac acgattgccc gacggattca tgagatgtca ggccgcaaag
     4561 ggcgcctggt ggcgatgaat tgcgcggcca ttccggagtc cctcgccgag agcgagttat
     4621 tcggcgtggt cagcggtgcc tacaccggcg ctgatcgctc cagagtcggt tatgtcgaag
     4681 cggcgcaggg cggcacgctg tacctggatg agatcgatag catgccgctg agcctgcaag
     4741 ccaaattgct gagggtgctg gaaacccgag cgcttgaacg gctgggttcg acgtcgacga
     4801 tcaagctgga tatctgcgtg atcgcctccg cccaatgctc gctggacgac gccgtcgagc
     4861 gggggcagtt tcgtcgcgat ctgtattttc gcctgaacgt cctgacactc aagcttcctc
     4921 cgctacgtaa ccagtctgat cgcatagttc ccctgttcac acgttttacg gccgccgccg
     4981 cgagggagct cggtgttccc gttcccgatg tttgcccact gctgcacaaa gtgctgctgg
     5041 gccacgactg gcccggcaat atccgtgagc tcaaggcggc agccaaacgc catgtgctgg
     5101 gtttcccctt gctgggcgcc gagccgcagg gcgaagagca cttggcctgt gggctcaaat
     5161 cgcaattgcg agtgatcgaa aaagccctga ttcaggagtc gctcaagcgc cacgacaatt
     5221 gtgtggattc ggtaagcctg gaactggacg tgccacgccg tacgctctat cgacgcatca
     5281 aagaattgca gatctaataa tactagagcc aggcatcaaa taaaacgaaa ggctcagtcg
     5341 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac
     5401 actggctcac cttcgggtgg gcctttctgc gtttatatac tagaggccgg attatgtccg
     5461 ctgagtgggt cacggtcccg gatcagttcc cttgcgaagc tgaccgatgt ttttgtgcca
     5521 aaagctgttg tggcaaaaaa cggtttgcgc aaagttttgt attacaaaga atttcacatt
     5581 ttaaaatatc tttataaatc aatcagttat ttctattttc aagctggcac ggttattgct
     5641 atagggcttg tactactaga gattaaagag gagaaatact agatgcgtaa aggagaagaa
     5701 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     5761 ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac ccttaaattt
     5821 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttcggttat
     5881 ggtgttcaat gctttgcgag atacccagat catatgaaac agcatgactt tttcaagagt
     5941 gccatgcccg aaggttatgt acaggaaaga actatatttt tcaaagatga cgggaactac
     6001 aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     6061 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac
     6121 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa
     6181 attagacaca acattgaaga tggaagcgtt caactagcag accattatca acaaaatact
     6241 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac acaatctgcc
     6301 ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct
     6361 gctgggatta cacatggcat ggatgaacta tacaaaggcg gctccggcgg ctccggcggc
     6421 accggcggct ccggcggctc cgaaaacctg tattttcagg gtggcgctgc aaacgacgaa
     6481 aactacgctg ccgcagttta ataatactag agccaggcat caaataaaac gaaaggctca
     6541 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     6601 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat atactagtag cggccgctgc
     6661 a
//
