LOCUS       sequence-73351-e        7349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7349)
  AUTHORS   Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA
  TITLE     Genomic mining of prokaryotic repressors for orthogonal logic gates.
  JOURNAL   Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411.
  PUBMED    24316737
REFERENCE   2  (bases 1 to 7349)
  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: "Nat Chem 
            Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411."
COMMENT     Sequence Label: pRF-TetR
FEATURES             Location/Qualifiers
     source          1..7349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(3..8,9..24,25..31)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..56
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             91..714
                     /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(164..183)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      741..812
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      828..855
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    877..895
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             939..950
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(957..959,960..962,963..1676)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1002..1023)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1263..1282)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1610..1631
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1716..1733)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1886..1972
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2064..2091
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2110..2201
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2202..2270,2271..3062)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3104..3559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3191..3210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4390..4407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4524..5069)
                     /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(4604..4623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5141..5160
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6135..7217)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7179..7198
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt ttcacacagg aaaggcctcg atgtccagat tagataaaag taaagtgatt
      121 aacagcgcat tagagctgct taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc
      181 gcccagaagc taggtgtaga gcagcctaca ttgtattggc atgtaaaaaa taagcgggct
      241 ttgctcgacg ccttagccat tgagatgtta gataggcacc atactcactt ttgcccttta
      301 gaaggggaaa gctggcaaga ttttttacgt aataacgcta aaagttttag atgtgcttta
      361 ctaagtcatc gcgatggagc aaaagtacat ttaggtacac ggcctacaga aaaacagtat
      421 gaaactctcg aaaatcaatt agccttttta tgccaacaag gtttttcact agagaatgca
      481 ttatatgcac tcagcgctgt ggggcatttt actttaggtt gcgtattgga agatcaagag
      541 catcaagtcg ctaaagaaga aagggaaaca cctactactg atagtatgcc gccattatta
      601 cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc cagccttctt attcggcctt
      661 gaattgatca tatgcggatt agaaaaacaa cttaaatgtg aaagtgggtc ctaataacta
      721 gggcccatac ccccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
      781 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
      841 gtgggccttt ctgcgtttat acgacgtacg gtggaatccc tatcagtgat agagattgac
      901 atccctatca gtgatagata taatgagcac tactagagaa agaggagaaa tactagatgg
      961 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     1021 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1081 agctgaccct gaagttcatc tgcaccacag gcaagctgcc cgtgccctgg cccaccctcg
     1141 tgaccacctt cggctacggc ctgcaatgct tcgcccgcta ccccgaccac atgaagctgc
     1201 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1261 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1321 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1381 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1441 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1501 actaccagca gaacacccca atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1561 tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1621 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaataag
     1681 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
     1741 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     1801 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     1861 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1921 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     1981 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     2041 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     2101 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2161 aaccctgata aatgcttcaa taatattgaa aaaggaagac tatgagtatt caacatttcc
     2221 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2281 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2341 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2401 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2461 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2521 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2581 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2641 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2701 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2761 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2821 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2881 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2941 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3001 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3061 aactgtcaga ccaagtttac tcatatatac tttagattga tttacgcgcc ctgtagcggc
     3121 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3181 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3241 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3301 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3361 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3421 tgaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     3481 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3541 atattaacgt ttacaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3601 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3661 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3721 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3781 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3841 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3901 agtcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac
     3961 cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcgaat
     4021 ttgctttcga atttctgcca ttcatccgct tattatcact tattcaggcg tagcaccagg
     4081 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     4141 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaga cggcatgatg
     4201 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggc
     4261 tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc
     4321 atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata
     4381 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat
     4441 ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt
     4501 gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa
     4561 atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4621 ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
     4681 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
     4741 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
     4801 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
     4861 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
     4921 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
     4981 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
     5041 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
     5101 tgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5161 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5221 tctgctcatg tttgacagct tatcatcgat gcataatgtg cctgtcaaat ggacgaagca
     5281 gggattctgc aaaccctatg ctactccctc gagccgtcaa ttgtctgatt cgttaccaat
     5341 tattaatttt taaagtatgg gcaatcaatt gctcctgtta aaattgcttt agaaatactt
     5401 tggcagcggt ttgttgtatt gagtttcatt tgcgcattgg ttaaatggaa agtgacagta
     5461 cgctcactgc aacctaatat ttttgaaata tcccaagagc tttttccttc gcatgcccac
     5521 gctaaacatt ctttttctct tttggttaaa tcgttgtttg atttattatt tgctatattt
     5581 atttttcgat aattatcaac tagagaagga acaattaatg gtatgttcat acacgcatgt
     5641 aaaaataaac tatctatata gttgtctttt tctgaatgtg caaaactaag cattccgaag
     5701 ccattgttag ccgtatgaat agggaaacta aacccagtga taagacctga tgttttcgct
     5761 tctttaatta catttggaga ttttttattt acagcattgt tttcaaatat attccaatta
     5821 attggtgaat gattggagtt agaataatct actataggat catattttat taaattagcg
     5881 tcatcataat attgcctcca ttttttaggg taattatcta ggattgaaat atcagattta
     5941 accatagaat gaggataaat gatcgcgagt aaataatatt cacaatgtac cattttagtc
     6001 atatcagata agcattgatt aatatcatta ttgcttctac aagctttaat tttattaatt
     6061 attctgtatg tgtcgtcggc atttatgttt ttcatcgagg cctttcctgt gtgacccgct
     6121 ggaataggat cttatcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6181 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6241 cttttcacca gtgagactgg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6301 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6361 aacggcggga tataacatga gctatcttcg gtatcgtcgt atcccactac cgagatatcc
     6421 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6481 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6541 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6601 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6661 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6721 ccgtcctcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6781 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6841 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6901 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6961 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     7021 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     7081 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7141 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7201 aacgttactg gtttcatatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7261 ataccgcgaa aggttttgcg ccattcgatg gcgcgccgct tcgtcaggcc acatagcttt
     7321 cttgttctga tcggaacgat cgttggctg
//
