LOCUS       sequence-199387-        4215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline inducible expression of GFPmut2 using bicistronic 
            autoregulation; moderate ribosome binding site; GFPmut2 ORF can be 
            replaced with gene of interest by isothermal assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4215)
  AUTHORS   Hensel Z
  TITLE     A plasmid-based Escherichia coli gene expression system with 
            cell-to-cell variation below the extrinsic noise limit.
  JOURNAL   PLoS One. 2017 Oct 30;12(10):e0187259. doi: 
            10.1371/journal.pone.0187259. eCollection 2017.
  PUBMED    29084263
REFERENCE   2  (bases 1 to 4215)
  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: "PLoS One.";
            date: "2017-10-30"; volume: "12(10):e0187259. doi"; pages: " 
            10.1371/journal.pone.0187259. eCollection 2017"
COMMENT     Sequence Label: pZH512
FEATURES             Location/Qualifiers
     source          1..4215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..19,20..25,26..42,43..48,49..74)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             104..817
                     /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(134..162)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     757..777
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             844..1467
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(917..936)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1512..1558
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1577..1593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1577..1593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1590..1612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1601..1617
                     /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)."
     promoter        complement(join(1625..1631,1632..1649,1650..1655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1670..1691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2231..2776)
                     /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(2311..2330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2967..3758,3759..3827))
                     /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     3590..3609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3828..3932)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4000..4018
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4056..4078)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4178..4197
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcagc
       61 aggacgcact gaccgaattc attaaagccg agaaaggtac cgcatgagta aaggagaaga
      121 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
      181 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
      241 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcgcgta
      301 tggtcttcaa tgctttgcga gatacccaga tcatatgaaa cagcatgact ttttcaagag
      361 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
      421 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
      481 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
      541 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
      601 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
      661 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
      721 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
      781 tgctgggatt acacatggca tggatgaact atacaaataa taatctagca ggaggatttc
      841 accatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
      901 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
      961 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     1021 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     1081 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     1141 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     1201 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     1261 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     1321 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     1381 caaggtgcag agccagcctt cttattcggc cttgaattga tcatctgcgg attagaaaaa
     1441 caacttaaat gtgaaagtgg gtcttaataa ctgcagcccg ggggatccca tggtacgcgt
     1501 gctagaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttataa
     1561 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1681 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggcgcgctag cggagtgtat
     1981 actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga
     2041 aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg
     2101 ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg
     2161 ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg
     2221 caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa
     2281 atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct
     2341 ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc
     2401 cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg
     2461 gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt
     2521 cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga
     2581 tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt
     2641 tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac
     2701 cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag
     2761 accaaaacga tctcaagaag atcatcttat taatcagata aaatatagat cttttctacg
     2821 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2881 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2941 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3001 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3061 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3121 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3181 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3241 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3301 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3361 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3421 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3481 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3541 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3601 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3661 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3721 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3781 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3841 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3901 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3961 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     4021 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4081 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4141 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     4201 agagtgcacc atatg
//
