LOCUS       sequence-199582-        4333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline inducible expression of GFPmut2 using autoregulation; 
            strong 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 4333)
  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 4333)
  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: pZH517
FEATURES             Location/Qualifiers
     source          1..4333
                     /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"
     RBS             84..95
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     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"
     promoter        join(874..892,893..898,899..915,916..921,922..947)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    874..892
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    899..917
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             962..1585
                     /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(1035..1054)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1630..1676
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1695..1711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1695..1711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1708..1730)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1719..1735
                     /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(1743..1749,1750..1767,1768..1773))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1788..1809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2349..2894)
                     /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(2429..2448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3085..3876,3877..3945))
                     /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     3708..3727
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3946..4050)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4118..4136
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4174..4196)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4296..4315
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacatcagc
       61 aggacgcact gaccgaattc attaaagagg 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 tattccctat cagtgataga gattgacatc
      901 cctatcagtg atagagatac tgagcacatc agcaggacgc actgaccagg aggatttcac
      961 catgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt
     1021 cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag agcagcctac
     1081 attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt
     1141 agataggcac catactcact tttgcccttt agaaggggaa agctggcaag attttttacg
     1201 taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca
     1261 tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt
     1321 atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg tggggcattt
     1381 tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac
     1441 acctactact gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca
     1501 aggtgcagag ccagccttct tattcggcct tgaattgatc atctgcggat tagaaaaaca
     1561 acttaaatgt gaaagtgggt cttaataact gcagcccggg ggatcccatg gtacgcgtgc
     1621 tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttataagc
     1681 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1741 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1801 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1861 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     1921 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1981 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2041 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg cgcgctagcg gagtgtatac
     2101 tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa
     2161 aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct
     2221 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg
     2281 cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca
     2341 aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat
     2401 cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc
     2461 ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg
     2521 cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga
     2581 ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct
     2641 tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt
     2701 tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg
     2761 gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct
     2821 tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac
     2881 caaaacgatc tcaagaagat catcttatta atcagataaa atatagatct tttctacggg
     2941 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3001 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3061 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3121 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3181 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3241 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3301 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3361 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3421 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3481 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3541 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3601 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3661 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     3721 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     3781 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     3841 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     3901 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3961 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4021 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4081 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     4141 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     4201 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     4261 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     4321 agtgcaccat atg
//
