LOCUS       sequence-95189-e        7224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Triple split (at 67, 601-SZ).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7224)
  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 7224)
  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_14
FEATURES             Location/Qualifiers
     source          1..7224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             206..217
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             1986..1997
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3043..3074
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     rep_origin      3585..4129
                     /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     4030..4049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4246..4263
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             4838..5629
                     /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(5756..5785)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(5848..6930)
                     /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     6892..6911
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7065..7070,7071..7086,7087..7093)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    7099..7118
                     /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        7134..7183
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             7207..7218
                     /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 tctacaagaa agcatttatg caagttgtcg aggctgacat
      601 gctctctaag ggtctactcg gtggcgaggc gtggtcttcg tggcataagg aagactctat
      661 tcatgtagga gtacgctgca tcgagatgct cattgagtca accggaatgg ttagcttaca
      721 ccgccaaaat gctggcgtag taggtcaaga ctctgagact atcgaactcg cacctgaata
      781 cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc atctctccga tgttccaacc
      841 ttgcgtagtt cctcctaagc cgtggactgg cattactggt ggtggctatt gggctaacgg
      901 tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa gcactgatgc gctacgaaga
      961 cgtttacatg cctgaggtgt acaaagcgat taacattgcg caaaacaccg catggaaaat
     1021 caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag tggaagcatt gtccggtcga
     1081 ggacatccct gcgattgagc gtgaagaact cccgatgaaa ccggaagaca tcgacatgaa
     1141 tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct gtgtaccgca aggacaaggc
     1201 tcgcaagtct cgccgtatca gccttgagtt catgcttgag caagccaata agtttgctaa
     1261 ccataaggcc atctggttcc cttacaacat ggactggcgc ggtcgtgttt acgctgtgtc
     1321 aatgttcaac ccgcaaggta acgatatgac caaaggactg cttacgctgg cgaaaggtaa
     1381 accaatcggt aaggaaggtt actactggct gaaaatccac ggtgcaaact gtgcgggtgt
     1441 cgacaaggtt ccgttccctg agcgcatcaa gttcattgag gaaaaccacg agaacatcat
     1501 ggcttgcgct aagtctccac tggagaacac ttggtgggct gagcaagatt ctccgttctg
     1561 cttccttgcg ttctgctttg agtacgctgg ggtacagcac cacggcctga gctataactg
     1621 ctcccttccg ctggcgtttg acgggtcttg ctctggcatc cagcacttct ccgcgatgct
     1681 ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct agtgaaaccg ttcaggacat
     1741 ctacgggatt gttgctaaga aagtcaacga gattctacaa gcagacgcaa tcaatgggac
     1801 cgataacgaa gtagttaccg tgaccgatga gaacggaggt tcaggtggtg gatccaacga
     1861 aaaagaagaa ctgaaatcca aaaaagcgga actgcgcaac cgtatcgaac agctgaaaca
     1921 gaaacgtgaa caactgaagc agaaaatcgc gaacctgcgt aaagaaatcg aagcttacaa
     1981 ataataaaga ggagaaatac tagatgagca tcgcggcgac cctggagaac gatctggcgc
     2041 gtctggaaaa cgaaaacgct cgtctcgaaa aagacatcgc gaacctggaa cgtgacctgg
     2101 cgaaactgga gcgtgaagaa gcgtacttcg gaggttcagg tggtaagaac actggtgaaa
     2161 tctctgagaa agtcaagctg ggcactaagg cactggctgg tcaatggctg gcttacggtg
     2221 ttactcgcag tgtgactaag agttcagtca tgacgctggc ttacgggtcc aaagagttcg
     2281 gcttccgtca acaagtgctg gaagatacca ttcagccagc tattgattcc ggcaagggtc
     2341 tgatgttcac tcagccgaat caggctgctg gatacatggc taagctgatt tgggaatctg
     2401 tgagcgtgac ggtggtagct gcggttgaag caatgaactg gcttaagtct gctgctaagc
     2461 tgctggctgc tgaggtcaaa gataagaaga ctggagagat tcttcgcaag cgttgcgctg
     2521 tgcattgggt aactcctgat ggtttccctg tgtggcagga atacaagaag cctattcaga
     2581 cgcgcttgaa cctgatgttc ctcggtcagt tccgcttaca gcctaccatt aacaccaaca
     2641 aagatagcga gattgatgca cacaaacagg agtctggtat cgctcctaac tttgtacaca
     2701 gccaagacgg tagccacctt cgtaagactg tagtgtgggc acacgagaag tacggaatcg
     2761 aatcttttgc actgattcac gactccttcg gtacgattcc ggctgacgct gcgaacctgt
     2821 tcaaagcagt gcgcgaaact atggttgaca catatgagtc ttgtgatgta ctggctgatt
     2881 tctacgacca gttcgctgac cagttgcacg agtctcaatt ggacaaaatg ccagcacttc
     2941 cggctaaagg taacttgaac ctccgtgaca tcttagagtc ggacttcgcg ttcgcgtaac
     3001 gctgcaggag tcactaaggg ttagttagtt agattagcag aaagtcaaaa gcctccgacc
     3061 ggaggctttt gactaaaact tcccttgggg ttatcattgg ggctcactca aaggcggtaa
     3121 tcagataaaa aaaatcctta gctttcgcta aggatgattt ctgctagaga tggaatagac
     3181 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3241 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3301 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3361 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3421 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3481 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3541 ttttcgttcc actgagcgtc agacccctta ataagatgat cttcttgaga tcgttttggt
     3601 ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag
     3661 gttctctgag ctaccaactc tttgaaccga ggtaactggc ttggaggagc gcagtcacca
     3721 aaacttgtcc tttcagttta gccttaaccg gcgcatgact tcaagactaa ctcctctaaa
     3781 tcaattacca gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt tggactcaag
     3841 acgatagtta ccggataagg cgcagcggtc ggactgaacg gggggttcgt gcatacagtc
     3901 cagcttggag cgaactgcct acccggaact gagtgtcagg cgtggaatga gacaaacgcg
     3961 gccataacag cggaatgaca ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg
     4021 agccgccagg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc accactgatt
     4081 tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa acggctttgc
     4141 cgcggccctc tcacttccct gttaagtatc ttcctggcat cttccaggaa atctccgccc
     4201 cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc
     4261 gaggaagcgg aatatatcct gtatcacata ttctgctgac gcaccggtgc agcctttttt
     4321 ctcctgccac atgaagcact tcactgacac cctcatcagt gccaacatag taagccagta
     4381 tacactccgc tagcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc
     4441 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
     4501 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc
     4561 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagcca
     4621 cgttgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     4681 aataaaactg tctgcttaca taaacagtaa tacaaggggt gtttactaga ggttgatcgg
     4741 gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
     4801 tgagttatcg agattttcag gagctaagga agctaaaatg agggaagcgg tgatcgccga
     4861 agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt
     4921 gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat
     4981 tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa
     5041 cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt
     5101 caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca
     5161 atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga
     5221 cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc
     5281 agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga
     5341 aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct
     5401 tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc
     5461 tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag
     5521 acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga
     5581 atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataat actagctccg
     5641 gcaaaaaaac gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac
     5701 ttcgcgtttg ccacctgacg tctaagaaaa ggaatattca gcaatttgcc cgtgccgaag
     5761 aaaggcccac ccgtgaaggt gagccagtga gttgattgct acgtaatgtc ggccaattcg
     5821 cgctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5881 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5941 cagggtggtt tttcttttca ccagtgagac tggcaacagc tgattgccct tcaccgcctg
     6001 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     6061 tttgatggtg gttaacggcg ggatataaca tgagctatct tcggtatcgt cgtatcccac
     6121 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     6181 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     6241 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     6301 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     6361 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     6421 gcccagtcgc gtaccgtcct catgggagaa aataatactg ttgatgggtg tctggtcaga
     6481 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     6541 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6601 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6661 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6721 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6781 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6841 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6901 tgcgacatcg tataacgtta ctggtttcat attcaccacc ctgaattgac tctcttccgg
     6961 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggcgcgcc gcttcgtcag
     7021 gccacatagc tttcttgttc tgatcggaac gatcgttggc tgtgttgaca attaatcatc
     7081 ggctcgtata atgtgtggaa ttgtgagcgc tcacaattct atggactatg tttctgtcac
     7141 cggatgtgct ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt
     7201 gtttaaaaag aggagaaata ctag
//
