LOCUS       sequence-445465-        4506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP in response to Isoeugenol.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4506)
  AUTHORS   d'Oelsnitz S, Love JD, Ellington AD, Ross D
  TITLE     Ligify: Automated Genome Mining for Ligand-Inducible Transcription 
            Factors.
  JOURNAL   ACS Synth Biol. 2024 Aug 16;13(8):2577-2586. doi: 
            10.1021/acssynbio.4c00372. Epub 2024 Jul 19.
  PUBMED    39029917
REFERENCE   2  (bases 1 to 4506)
  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: 
            "10.1021/acssynbio.4c00372"; journalName: "ACS Synth Biol"; date: 
            "2024-08-16- 16"; volume: "13"; issue: "8"; pages: "2577-2586"
COMMENT     Sequence Label: pLigify_Isoeugenol
FEATURES             Location/Qualifiers
     source          1..4506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..30
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     misc_RNA        459..508
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     primer_bind     complement(1722..1738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1722..1738)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1735..1757)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(1918..2463)
                     /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(1998..2017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2574..3389)
                     /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(2574..3389)
                     /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     3297..3316
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3381..3400
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     3510..3526
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3510..3526
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      3527..3573
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3687..3709
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3701..3718
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3702..3718
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(3853..4506,1..60))
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3893..3913)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 accatctaat tcaacaagaa ttgggacaac tccagtgaaa agttcttctc ctttactcat
       61 atataccccc ttattctccc gtattaaaca aaattatttg tagaggcccc atttcgtcct
      121 tttggactca tcaggggtgg tacacaccac cctatggggc tggcacggtc tcctttgttg
      181 ttctgaatgg cctaaatgcg cggcttgccg ggttggagtt tatgtttagg actgaccgga
      241 tttcatgtgt gccggtgaag tgaggatgtc tgtgagtgca atggtggcgg tattgaaaat
      301 gggccgaggc tggcctattg ttgaggattt caagatttct tgcaaaaaaa aaccccgccg
      361 aagcggggtt ttttttttta caggggaaac gaaaatatat ttttcaaaag tatcgtttac
      421 cgctagctca gtcctaggta caatgagcac agtggcagct gtcaccggat gtgctttccg
      481 gtctgatgag tccgtgagga cgaaacagcc tctacaaata attttgttta agggcccaag
      541 ttcacttaaa aaggagatca acaatgaaag caattttcgt actgaaacat cttaatcatg
      601 caccggtgtt ttaaatatga ccaccatccg ctggcgccgt atgtctattc attcggagcg
      661 catcactctt gcagatagcc ctttacattg ggcacataca ttgaatggtt ctatgcgtac
      721 ccattttgaa gtacaacgct tagagcgcgg gcgcggtgct tacttggcgc gcagtcgctt
      781 cggtgctggg gaattgtact cggccatcgc cccttcccag gtcttacgtc acttcaatga
      841 tcaacgtaac gccaacgaag cggaacactc ctacctgatt cagatccgct cgggggcctt
      901 aggcgtggcc agtggaggtc gtaaagttat cttagcaaat ggcgattgta gtattgtcga
      961 ttcgcgccaa gacttcacgt tgtcgtccaa ctcatctact caaggggtcg tgatccgttt
     1021 tcctgtgagt tggttgggtg cctgggtatc caatcccgaa gatcttattg cccgtcgcgt
     1081 tgacgcagaa attggatggg ggcgcgcgtt gtctgctagt gtctccaact tagacccgct
     1141 gcgtatcgac gacttaggaa gtaacgtcaa ttcgattgca gagcatgtcg ctatgttaat
     1201 ttcacttgct agctcggctg tgagttcgga ggacggggga gtagccttac gcaaaatgcg
     1261 tgaggtcaag cgcgtcttag aacagtcatt tgccgatgcg aatcttgaac cagaaagtgt
     1321 ttcaagccag cttggtatta gtaagcgcta cttacactac gtttttgctg cgtgtggaac
     1381 aacttttggc cgtgaattat tggagatccg cttgggaaag gcttaccgta tgctgtgtgc
     1441 gacttcagga agtggggctg ttcttaaggt ggctatgtcc agtgggttta gcgattcgag
     1501 ccacttctcc aaaaaattca aggaacgtta cggagtgtct cctgtgtcgt tagtccgcca
     1561 agcttaataa tcactttcag ccaaaaaact taagaccgcc ggtcttgtcc actaccttgc
     1621 agtaatgcgg tggacaggat cggcggtttt cttttctctt ctcaaaggct aggtggaggc
     1681 tcagtgatga taagtctgcg atggtggatg catgtgtcat ggtcatagct gtttcctgtg
     1741 tgaaattgtt atccgctcag agggcacaat cctattccgc gctatccgac aatctccaag
     1801 acattaggtg gagttcagtt cggcgagcgg aaatggctta cgaacggggc ggagatttcc
     1861 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt
     1921 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg
     1981 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc
     2041 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca
     2101 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg
     2161 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2221 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta
     2281 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct
     2341 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg
     2401 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct
     2461 caagaagatc atcttattaa gtctgacgct ctattcaaca aagccgccgt ccatgggtag
     2521 ggggcttcaa atcgtccgct ctgccagtgt tacaaccaat taacaaattc tgattagaaa
     2581 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     2641 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     2701 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2761 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2821 ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
     2881 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2941 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3001 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3061 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3121 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3181 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3241 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3301 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag
     3361 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     3421 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
     3481 tgagacacaa cgtggctttc ccccgccgct ctagaactag tggatccaaa taaaacgaaa
     3541 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcgcatt atacgagacg
     3601 tccaggttgg gatacctgaa acaaaaccca tcgtacggcc aaggaagtct ccaataactg
     3661 tgatccacca caagcgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtca
     3721 tgcataatcc gcacgcatct ggaataagga agtgccattc cgcctgacct ggaccaaaac
     3781 gaaaaaaggg gagcggtttc ccgctcccct cttttctgga atttggtacc gaggaatgaa
     3841 gcaggattat tatttgtata gttcatccat gccatgtgta atcccagcag ctgttacaaa
     3901 ctcaagaagg accatgtggt ctctcttttc gttgggatct ttcgaaaggg cagattgtgt
     3961 ggacaggtaa tggttgtctg gtaaaaggac agggccatcg ccaattggag tattttgttg
     4021 ataatggtct gctagttgaa cgcttccatc ttcaatgttg tgtctaattt tgaagttaac
     4081 tttgattcca ttcttttgtt tgtctgccat gatgtataca ttgtgtgagt tatagttgta
     4141 ttccaatttg tgtccaagaa tgtttccatc ttctttaaaa tcaatacctt ttaactcgat
     4201 tctattaaca agggtatcac cttcaaattt gacttcagca cgtgtcttgt agttcccgtc
     4261 atctttgaaa aatatagttc tttcctgtac ataaccttcg ggcatggcac tcttgaaaaa
     4321 gtcatgctgt ttcatatgat ctgggtatct cgcaaagcat tgaagaccat acgcgaaagt
     4381 agtgacaagt gttggccatg gaacaggtag ttttccagta gtgcaaataa atttaagggt
     4441 aagttttccg tatgttgcat caccttcacc ctctccactg acagaaaatt tgtgcccatt
     4501 aacatc
//
