LOCUS       sequence-431908-        4234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Theo-3c riboswitch-regulated GFP reporter. The gene is inserted into
            downstream of T7 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4234)
  AUTHORS   Ishii Y, Fukunaga K, Cooney A, Yokobayashi Y, Matsuura T
  TITLE     Switchable and orthogonal gene expression control inside artificial 
            cells by synthetic riboswitches.
  JOURNAL   Chem Commun (Camb). 2024 Jun 4;60(46):5972-5975. doi: 
            10.1039/d4cc00965g.
  PUBMED    38767578
REFERENCE   2  (bases 1 to 4234)
  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"; journalName: 
            "Chem Commun (Camb)"; date: "2024-06-4- 4"; volume: "60"; issue: 
            "46"; pages: "5972-5975"
COMMENT     Sequence Label: pIVEX2.3-Theo-3c-EGFP
FEATURES             Location/Qualifiers
     source          1..4234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     328..347
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     581..598
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    715..736
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(751..756,757..774,775..781)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    789..805
                     /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)."
     primer_bind     794..816
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     813..829
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     813..829
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        join(850..855,856..872,873..878)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(1244..1291)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1287..1305
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(1396..2109,2110..2112,2113..2115))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1399..2109,2110..2112,2113..2115))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1441..1462)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1790..1809
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2049..2070
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     RBS             2142..2150
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(2205..2224)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2206..2224)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2308..2327)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2447..2464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2447..2463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2456..2478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2672..2691)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2791..2813
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2851..2869)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2937..3041
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3042..3110,3111..3902)
                     /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     complement(3260..3279)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4073..4234,1..427)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
       61 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      121 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      181 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      241 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      301 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      361 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      421 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      481 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      541 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      601 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      661 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      721 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      781 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
      841 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
      901 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      961 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
     1021 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata
     1081 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg
     1141 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac
     1201 cacacccgtc ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     1261 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     1321 cagcttcctt tcgggctttg ttagcagccg gatccactag taacggccgc cagtgtgctg
     1381 gaattcgccc ttttattact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     1441 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     1501 ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     1561 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     1621 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     1681 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     1741 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     1801 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     1861 gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt
     1921 cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     1981 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     2041 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2101 gcccttgctc accatggata tacgagccga aagcattttt tacctcctta attcgataac
     2161 cgggtcgcat caaagagggc tgccaagggc ctttcggctg gtatccctat agtgagtcgt
     2221 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     2281 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     2341 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     2401 tggccggggg actgttgggc gccatctcct tgcatgcaag cttggcactg gccgtcgttt
     2461 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     2521 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     2581 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     2641 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2701 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     2761 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     2821 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     2881 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     2941 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3001 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3061 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3121 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3181 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3241 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3301 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3361 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3421 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3481 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3541 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3601 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3661 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3721 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3781 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3841 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3901 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3961 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4021 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4081 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4141 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4201 cgcagatacc aaatactgtt cttctagtgt agcc
//
