LOCUS       sequence-210286-        4304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LacIAM + PTac-YFP Reporter. IPTG sensor for use in non-Marionette 
            strain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4304)
  AUTHORS   Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA
  TITLE     Escherichia coli ""Marionette"" strains with 12 highly optimized 
            small-molecule sensors.
  JOURNAL   Nat Chem Biol. 2018 Nov 26. pii: 10.1038/s41589-018-0168-3. doi: 
            10.1038/s41589-018-0168-3.
  PUBMED    30478458
REFERENCE   2  (bases 1 to 4304)
  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 Chem 
            Biol. 2018 Nov 26. pii: 10.1038/s41589-018-0168-3. doi: 
            10.1038/s41589-018-0168-3."
COMMENT     Sequence Label: pAJM.336
FEATURES             Location/Qualifiers
     source          1..4304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..50
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(100..102,103..105,106..819)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(145..166)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(406..425)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     753..774
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             967..2049
                     /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="MKPVTLYDVAEYAGVSYMTVSRVVNQASHVSAKTREKVEAAMVEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIYSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(986..1005)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     primer_bind     complement(2241..2260)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(2625..3170)
                     /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(2705..2724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3259..4074)
                     /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(3259..4074)
                     /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     3982..4001
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4066..4085
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(4249..4254,4255..4270,4271..4277)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4283..4302
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
ORIGIN
        1 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag cctctacaaa
       61 taattttgtt taatactaga gaaagagggg aaatactaga tggtgagcaa gggcgaggag
      121 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
      181 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
      241 atctgcacca caggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
      301 ggcctgcaat gcttcgcccg ctaccccgac cacatgaagc tgcacgactt cttcaagtcc
      361 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
      421 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
      481 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
      541 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
      601 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
      661 ccaatcggcg acggccccgt gctgctgccc gacaaccact accttagcta ccagtccgcc
      721 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
      781 gccgggatca ctctcggcat ggacgagctg tacaagtaac tcggtaccaa attccagaaa
      841 agaggcctcc cgaaaggggg gccttttttc gttttggtcc aatggcggcg cgccatcgaa
      901 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
      961 gtgaatatga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatatg
     1021 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     1081 gaagcggcga tggtggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     1141 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     1201 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     1261 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     1321 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     1381 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     1441 attatttact cccatgagga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     1501 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     1561 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     1621 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     1681 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     1741 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagat
     1801 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     1861 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     1921 ttgccagtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct
     1981 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     2041 gggcagtgat aaggatccta attggtaacg aatcagacaa ttgacggctc gagggagtag
     2101 catagggttt gcagaatccc tgcttcgtcc atttgacagg cacattatgc atcgatgata
     2161 agctgtcaaa catgagcaga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     2221 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     2281 ccacttcggg ctcatgagca aatattttat ctgaggtgct tcctcgctca ctgactcgct
     2341 gcacgaggca gacctcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg
     2401 agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca
     2461 gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt
     2521 tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag
     2581 gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc
     2641 ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     2701 ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc
     2761 tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac
     2821 actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca
     2881 gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc
     2941 aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat
     3001 gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt
     3061 tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg
     3121 ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc
     3181 ttattaaggg gtctgacgct cagtggaacg aaaaatcaat ctaaagtata tatgagtaaa
     3241 cttggtctga cagttacctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     3301 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     3361 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     3421 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     3481 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca
     3541 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     3601 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     3661 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     3721 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     3781 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     3841 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     3901 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     3961 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4021 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     4081 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     4141 ttgtgcaatg tacatcagag attttgagac acaaccaatt attgaaggcc tccctaacgg
     4201 ggggcctttt tttgtttctg gtctcccgct taacgatcgt tggctgtgtt gacaattaat
     4261 catcggctcg tataatgtgt ggaattgtga gcgctcacaa ttag
//
