LOCUS       sequence-95192-e        7255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Quad split (at 61, 179, 601-SZ).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Segall-Shapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA
  TITLE     A 'resource allocator' for transcription based on a highly 
            fragmented T7 RNA polymerase.
  JOURNAL   Mol Syst Biol. 2014 Jul 30;10(7):742.
  PUBMED    25080493
REFERENCE   2  (bases 1 to 7255)
  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: "Mol Syst 
            Biol."; date: "2014-07-30"; volume: "10(7)"; pages: "742"
COMMENT     Sequence Label: pTHSSd_18
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             206..217
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             573..584
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2017..2028
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3074..3105
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     rep_origin      3616..4160
                     /direction=RIGHT
                     /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     4061..4080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4277..4294
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             4869..5660
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     terminator      complement(5787..5816)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(5879..6961)
                     /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     6923..6942
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7096..7101,7102..7117,7118..7124)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    7130..7149
                     /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."
     misc_RNA        7165..7214
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             7238..7249
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg
       61 ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag
      121 catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa
      181 gctggtgagg ttgcggataa ctaataaaga ggagaaatac tagatgaata acgctgccgc
      241 caagcctctc atcactaccc tactccctaa gatgattgca cgcatcaacg actggtttga
      301 ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc cagttcctgc aagaaatcaa
      361 gccggaagcc gtagcgtaca tcaccattaa gaccactctg gcttgcctaa ccagtgctga
      421 caatacaacc gttcaggctg tagcaagcgc aatcggtcgg gccattgagg acgaggctcg
      481 cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag aaaaacgttg aggaacaact
      541 caacaagcgc gtagggcacg tctacaagta ataaagagga gaaatactag atgaacaaga
      601 aagcatttat gcaagttgtc gaggctgaca tgctctctaa gggtctactc ggtggcgagg
      661 cgtggtcttc gtggcataag gaagactcta ttcatgtagg agtacgctgc atcgagatgc
      721 tcattgagtc aaccggaatg gttagcttac accgccaaaa tgctggcgta gtaggtcaag
      781 actctgagac tatcgaactc gcacctgaat acgctgaggc tatcgcaacc cgtgcaggtg
      841 cgctggctgg catctctccg atgttccaac cttgcgtagt tcctcctaag ccgtggactg
      901 gcattactgg tggtggctat tgggctaacg gtcgtcgtcc tctggcgctg gtgcgtactc
      961 acagtaagaa agcactgatg cgctacgaag acgtttacat gcctgaggtg tacaaagcga
     1021 ttaacattgc gcaaaacacc gcatggaaaa tcaacaagaa agtcctagcg gtcgccaacg
     1081 taatcaccaa gtggaagcat tgtccggtcg aggacatccc tgcgattgag cgtgaagaac
     1141 tcccgatgaa accggaagac atcgacatga atcctgaggc tctcaccgcg tggaaacgtg
     1201 ctgccgctgc tgtgtaccgc aaggacaagg ctcgcaagtc tcgccgtatc agccttgagt
     1261 tcatgcttga gcaagccaat aagtttgcta accataaggc catctggttc ccttacaaca
     1321 tggactggcg cggtcgtgtt tacgctgtgt caatgttcaa cccgcaaggt aacgatatga
     1381 ccaaaggact gcttacgctg gcgaaaggta aaccaatcgg taaggaaggt tactactggc
     1441 tgaaaatcca cggtgcaaac tgtgcgggtg tcgacaaggt tccgttccct gagcgcatca
     1501 agttcattga ggaaaaccac gagaacatca tggcttgcgc taagtctcca ctggagaaca
     1561 cttggtgggc tgagcaagat tctccgttct gcttccttgc gttctgcttt gagtacgctg
     1621 gggtacagca ccacggcctg agctataact gctcccttcc gctggcgttt gacgggtctt
     1681 gctctggcat ccagcacttc tccgcgatgc tccgagatga ggtaggtggt cgcgcggtta
     1741 acttgcttcc tagtgaaacc gttcaggaca tctacgggat tgttgctaag aaagtcaacg
     1801 agattctaca agcagacgca atcaatggga ccgataacga agtagttacc gtgaccgatg
     1861 agaacggagg ttcaggtggt ggatccaacg aaaaagaaga actgaaatcc aaaaaagcgg
     1921 aactgcgcaa ccgtatcgaa cagctgaaac agaaacgtga acaactgaag cagaaaatcg
     1981 cgaacctgcg taaagaaatc gaagcttaca aataataaag aggagaaata ctagatgagc
     2041 atcgcggcga ccctggagaa cgatctggcg cgtctggaaa acgaaaacgc tcgtctcgaa
     2101 aaagacatcg cgaacctgga acgtgacctg gcgaaactgg agcgtgaaga agcgtacttc
     2161 ggaggttcag gtggtaagaa cactggtgaa atctctgaga aagtcaagct gggcactaag
     2221 gcactggctg gtcaatggct ggcttacggt gttactcgca gtgtgactaa gagttcagtc
     2281 atgacgctgg cttacgggtc caaagagttc ggcttccgtc aacaagtgct ggaagatacc
     2341 attcagccag ctattgattc cggcaagggt ctgatgttca ctcagccgaa tcaggctgct
     2401 ggatacatgg ctaagctgat ttgggaatct gtgagcgtga cggtggtagc tgcggttgaa
     2461 gcaatgaact ggcttaagtc tgctgctaag ctgctggctg ctgaggtcaa agataagaag
     2521 actggagaga ttcttcgcaa gcgttgcgct gtgcattggg taactcctga tggtttccct
     2581 gtgtggcagg aatacaagaa gcctattcag acgcgcttga acctgatgtt cctcggtcag
     2641 ttccgcttac agcctaccat taacaccaac aaagatagcg agattgatgc acacaaacag
     2701 gagtctggta tcgctcctaa ctttgtacac agccaagacg gtagccacct tcgtaagact
     2761 gtagtgtggg cacacgagaa gtacggaatc gaatcttttg cactgattca cgactccttc
     2821 ggtacgattc cggctgacgc tgcgaacctg ttcaaagcag tgcgcgaaac tatggttgac
     2881 acatatgagt cttgtgatgt actggctgat ttctacgacc agttcgctga ccagttgcac
     2941 gagtctcaat tggacaaaat gccagcactt ccggctaaag gtaacttgaa cctccgtgac
     3001 atcttagagt cggacttcgc gttcgcgtaa cgctgcagga gtcactaagg gttagttagt
     3061 tagattagca gaaagtcaaa agcctccgac cggaggcttt tgactaaaac ttcccttggg
     3121 gttatcattg gggctcactc aaaggcggta atcagataaa aaaaatcctt agctttcgct
     3181 aaggatgatt tctgctagag atggaataga ctggatggag gcggataaag ttgcaggacc
     3241 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3301 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3361 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3421 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagacccctt
     3601 aataagatga tcttcttgag atcgttttgg tctgcgcgta atctcttgct ctgaaaacga
     3661 aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga gctaccaact ctttgaaccg
     3721 aggtaactgg cttggaggag cgcagtcacc aaaacttgtc ctttcagttt agccttaacc
     3781 ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg
     3841 cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3901 cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac
     3961 tgagtgtcag gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac
     4021 cgaaaggcag gaacaggaga gcgcacgagg gagccgccag gggaaacgcc tggtatcttt
     4081 atagtcctgt cgggtttcgc caccactgat ttgagcgtca gatttcgtga tgcttgtcag
     4141 gggggcggag cctatggaaa aacggctttg ccgcggccct ctcacttccc tgttaagtat
     4201 cttcctggca tcttccagga aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag
     4261 tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg gaatatatcc tgtatcacat
     4321 attctgctga cgcaccggtg cagccttttt tctcctgcca catgaagcac ttcactgaca
     4381 ccctcatcag tgccaacata gtaagccagt atacactccg ctagcgctga ggtctgcctc
     4441 gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc agccagaaag
     4501 tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg attttgaact
     4561 tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tccttcaact
     4621 cagcaaaagt tcgatttatt caacaaagcc acgttgtgtc tcaaaatctc tgatgttaca
     4681 ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta
     4741 atacaagggg tgtttactag aggttgatcg ggcacgtaag aggttccaac tttcaccata
     4801 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
     4861 aagctaaaat gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
     4921 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
     4981 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
     5041 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
     5101 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
     5161 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
     5221 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
     5281 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
     5341 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
     5401 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
     5461 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
     5521 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
     5581 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
     5641 ccaaggtagt cggcaaataa tactagctcc ggcaaaaaaa cgggcaaggt gtcaccaccc
     5701 tgcccttttt ctttaaaacc gaaaagatta cttcgcgttt gccacctgac gtctaagaaa
     5761 aggaatattc agcaatttgc ccgtgccgaa gaaaggccca cccgtgaagg tgagccagtg
     5821 agttgattgc tacgtaatgt cggccaattc gcgctaactt acattaattg cgttgcgctc
     5881 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5941 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6001 ctggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6061 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6121 atgagctatc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     6181 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6241 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6301 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6361 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6421 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtcc tcatgggaga
     6481 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6541 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6601 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6661 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6721 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6781 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6841 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6901 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6961 tattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7021 tgcgccattc gatggcgcgc cgcttcgtca ggccacatag ctttcttgtt ctgatcggaa
     7081 cgatcgttgg ctgtgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg
     7141 ctcacaattc tatggactat gtttctgtca ccggatgtgc tttccggtct gatgagtccg
     7201 tgaggacgaa acagcctcta caaataattt tgtttaaaaa gaggagaaat actag
//
