LOCUS       sequence-199391-        4403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline inducible expression of GFPmut2 with constitutive TetR;
            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 4403)
  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 4403)
  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: pZH522
FEATURES             Location/Qualifiers
     source          1..4403
                     /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"
     terminator      complement(830..873)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1032..1655
                     /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(1105..1124)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1700..1746
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1765..1781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1765..1781)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1778..1800)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1789..1805
                     /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(1813..1819,1820..1837,1838..1843))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1858..1879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2419..2964)
                     /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(2499..2518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3155..3946,3947..4015))
                     /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     3778..3797
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4016..4120)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4188..4206
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4244..4266)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4366..4385
                     /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 taaaacgaaa
      841 ggctcagtcg aaagactggg cctttcgttt tatcacagct aacaccacgt cgtccctatc
      901 tgctgcccta ggtctatgag tggttgctgg ataactttac gggcatgcat aaggctcgta
      961 atatatattc agggagacca caacggtttc cctctacaaa taattttgtt taactttagg
     1021 aggatttcac catgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc
     1081 ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag
     1141 agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca
     1201 ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa agctggcaag
     1261 attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag
     1321 caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat
     1381 tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg
     1441 tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag
     1501 aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct atcgaattat
     1561 ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc atctgcggat
     1621 tagaaaaaca acttaaatgt gaaagtgggt cttaataact gcagcccggg ggatcccatg
     1681 gtacgcgtgc tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1741 ttttataagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1801 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1861 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1921 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1981 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2041 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2101 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg cgcgctagcg
     2161 gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg
     2221 gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg
     2281 cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt
     2341 acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag
     2401 ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg
     2461 acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2521 tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct
     2581 gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct
     2641 ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta
     2701 actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg
     2761 gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg
     2821 acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct
     2881 cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt
     2941 acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa atatagatct
     3001 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3061 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3121 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3181 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3241 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3301 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3361 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3421 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3481 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3541 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3601 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3661 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3721 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3781 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3841 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3901 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3961 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4021 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4081 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4141 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4201 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4261 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4321 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     4381 ttgtactgag agtgcaccat atg
//
