LOCUS       sequence-384231-        9856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses an attenuated T7 RNAP under an isopropyl 
            beta-d-1-thiogalactopyranoside (IPTG) inducible control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9856)
  AUTHORS   Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, 
            Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA
  TITLE     Design of orthogonal genetic switches based on a crosstalk map of 
            sigmas, anti-sigmas, and promoters.
  JOURNAL   Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58.
  PUBMED    24169405
REFERENCE   2  (bases 1 to 9856)
  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: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pN565
FEATURES             Location/Qualifiers
     source          1..9856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    2776..2796
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      2832..2863
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             3725..4696
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein of plasmid pSa"
                     /label=RepA
                     /translation="MPKNNKAPGHRINEIIKTSLALEMEDAREAGLVGYMARCLVQATM
                     PHTDPKTSYFERTNGIVTLSIMGKPSIGLPYGSMPRTLLAWICTEAVRTKDPVLNLGRS
                     QSEFLQRLGMHTDGRYTATLRNQAQRLFSSMISLAGEQGNDFGIENVVIAKRAFLFWNP
                     KRPEDRALWDSTLTLTGDFFEEVTRSPVPIRIDYLHALRQSPLAMDIYTWLTYRVFLLR
                     AKGRPFVQIPWVALQAQFGSSYGSRARNSPELDDKARERAERAALASFKYNFKKRLREV
                     LIVYPEASDCIEDDGECLRIKSTRLHVTRAPGKGARIGPPPT"
     rep_origin      4733..5168
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     CDS             6843..7634
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRTDQLEEFVHYVKGEITKVVGK"
     terminator      complement(7911..7940)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(8043..9125)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVYDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     9087..9106
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9126..9203)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     promoter        join(9722..9727,9728..9743,9744..9750)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    9756..9775
                     /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."
ORIGIN
        1 gtgttgttta tcaagcctgc ggatctccgc gaaattgtga cttttccgct atttagcgat
       61 cttgttcagt gtggctttcc ttcaccggca gcagattacg ttgaacagcg catcgatctg
      121 ggtggcaaca cgattaacat cgctaagaac gacttctctg acatcgaact ggctgctatc
      181 ccgttcaaca ctctggctga ccattacggt gagcgtttag ctcgcgaaca gttggccctt
      241 gagcatgagt cttacgagat gggtgaagca cgcttccgca agatgtttga gcgtcaactt
      301 aaagctggtg aggttgcgga taacgctgcc gccaagcctc tcatcactac cctactccct
      361 aagatgattg cacgcatcaa cgactggttt gaggaagtga aagctaagcg cggcaagcgc
      421 ccgacagcct tccagttcct gcaagaaatc aagccggaag ccgtagcgta catcaccatt
      481 aagaccactc tggcttgcct aaccagtgct gacaatacaa ccgttcaggc tgtagcaagc
      541 gcaatcggtc gggccattga ggacgaggct cgcttcggtc gtatccgtga ccttgaagct
      601 aagcacttca agaaaaacgt tgaggaacaa ctcaacaagc gcgtagggca cgtctacaag
      661 aaagcattta tgcaagttgt cgaggctgac atgctctcta agggtctact cggtggcgag
      721 gcgtggtctt cgtggcataa ggaagactct attcatgtag gagtacgctg catcgagatg
      781 ctcattgagt caaccggaat ggttagctta caccgccaaa atgctggcgt agtaggtcaa
      841 gactctgaga ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac ccgtgcaggt
      901 gcgctggctg gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa gccgtggact
      961 ggcattactg gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct ggtgcgtact
     1021 cacagtaaga aagcactgat gcgctacgaa gacgtttaca tgcctgaggt gtacaaagcg
     1081 attaacattg cgcaaaacac cgcatggaaa atcaacaaga aagtcctagc ggtcgccaac
     1141 gtaatcacca agtggaagca ttgtccggtc gaggacatcc ctgcgattga gcgtgaagaa
     1201 ctcccgatga aaccggaaga catcgacatg aatcctgagg ctctcaccgc gtggaaacgt
     1261 gctgccgctg ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat cagccttgag
     1321 ttcatgcttg agcaagccaa taagtttgct aaccataagg ccatctggtt cccttacaac
     1381 atggactggc gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg taacgatatg
     1441 accaaaggac tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg ttactactgg
     1501 ctgaaaatcc acggtgcaaa ctgtgcgggt gtcgacaagg ttccgttccc tgagcgcatc
     1561 aagttcattg aggaaaacca cgagaacatc atggcttgcg ctaagtctcc actggagaac
     1621 acttggtggg ctgagcaaga ttctccgttc tgcttccttg cgttctgctt tgagtacgct
     1681 ggggtacagc accacggcct gagctataac tgctcccttc cgctggcgtt tgacgggtct
     1741 tgctctggca tccagcactt ctccgcgatg ctccgagatg aggtaggtgg tcgcgcggtt
     1801 aacttgcttc ctagtgaaac cgttcaggac atctacggga ttgttgctaa gaaagtcaac
     1861 gagattctac aagcagacgc aatcaatggg accgataacg aagtagttac cgtgaccgat
     1921 gagaacactg gtgaaatctc tgagaaagtc aagctgggca ctaaggcact ggctggtcaa
     1981 tggctggctt acggtgttac tcgcagtgtg actaagagtt cagtcatgac gctggcttac
     2041 gggtccaaag agttcggctt ccgtcaacaa gtgctggaag ataccattca gccagctatt
     2101 gattccggca agggtctgat gttcactcag ccgaatcagg ctgctggata catggctaag
     2161 ctgatttggg aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat gaactggctt
     2221 aagtctgctg ctaagctgct ggctgctgag gtcaaagata agaagactgg agagattctt
     2281 cgcaagcgtt gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg gcaggaatac
     2341 aagaagccta ttcagacgcg cttgaacctg atgttcctcg gtcagttccg cttacagcct
     2401 accattaaca ccaacaaaga tagcgagatt gatgcacaca aacaggagtc tggtatcgct
     2461 cctaactttg tacacagcca agacggtagc caccttcgta agactgtagt gtgggcacac
     2521 gagaagtacg gaatcgaatc ttttgcactg attcacgact ccttcggtac gattccggct
     2581 gacgctgcga acctgttcaa agcagtgcgc gaaactatgg ttgacacata tgagtcttgt
     2641 gatgtactgg ctgatttcta cgaccagttc gctgaccagt tgcacgagtc tcaattggac
     2701 aaaatgccag cacttccggc taaaggtaac ttgaacctcc gtgacatctt agagtcggac
     2761 ttcgcgttcg cgtaatacta gtagcggccg ctgcaggagt cactaagggt tagttagtta
     2821 gattagcaga aagtcaaaag cctccgaccg gaggcttttg actaaaactt cccttggggt
     2881 tatcattggg gctcactcaa aggcggtaat cagataaaaa aaatccttag ctttcgctaa
     2941 ggatgatttc tgctagagct gtcagaccag agctccaaac caacgtttta tctataccgc
     3001 tacagggtat ttaattccta tttaatctgc gctagaatga ggcatgttta accgaatctg
     3061 acgttttccc tgcaaatgcc aaaatactat gcctatctcc gggtttcgcg tgacggccaa
     3121 gacccggaaa accaaaaata cggtttgctc gaatacgcga acgccaaagg cttcgcgccg
     3181 ctacagatcg aggaagaaat tgccagcaga gcaaaggact ggcgcaagcg caagctcgga
     3241 gcaatcatcg aaaaggccga gcgtggcgac gtgctactga cgccggagat tacgcgcatt
     3301 gccggttccg ccctcgccgc cttggaaatt ctcaaagcgg cgagcgagcg cggcctaatc
     3361 gtccatgtga ccaaacagaa gatcatcatg gacggcagcc tacaaagcga catcatggca
     3421 accgtgcttg gcttggctgc acagatcgag cggcatttca ttcaggcacg taccaccgag
     3481 gcgctacaag tcgccagaga gcgcggcaag acgctcgggc gacccaaggg cagcaaatcg
     3541 agcgccttga agctggacag ccgtattgat gaagtacagg catacgtgaa ccttggcttg
     3601 ccgcaaagtc gcgcagccga gttgttaggc gtcagccctc acaccttgcg cctgttcatc
     3661 aaacgccgga acatcaaacc cacaaacact agaccaacca tcaccatgcc ggggagggaa
     3721 caacatgcct aagaacaaca aagcccccgg ccatcgtatc aacgagatca tcaagacgag
     3781 cctcgcgctc gaaatggagg atgcccgcga agctggctta gtcggctaca tggcccgttg
     3841 ccttgtgcaa gcgaccatgc cccacaccga ccccaagacc agctactttg agcgcaccaa
     3901 tggcatcgtc accttgtcga tcatgggcaa gccgagcatc ggcctgccct acggttctat
     3961 gccgcgcacc ttgcttgctt ggatatgcac cgaggccgtg cgaacgaaag accccgtgtt
     4021 gaaccttggc cggtcgcaat cggaatttct acaaaggctc ggaatgcaca ccgatggccg
     4081 ttacacggcc acccttcgca atcaggcgca acgcctgttt tcatccatga tttcgcttgc
     4141 cggcgagcaa ggcaatgact tcggcattga gaacgtcgtc attgccaagc gcgcttttct
     4201 attctggaat cccaagcggc cagaagatcg ggcgctatgg gatagcaccc tcaccctcac
     4261 aggcgatttc ttcgaggaag tcacccgctc accggttcct atccgaatcg actacctgca
     4321 tgccttgcgg cagtctccgc ttgcgatgga catttacacg tggctgacct atcgcgtgtt
     4381 cctgttgcgg gccaagggcc gccccttcgt gcaaatccct tgggtcgccc tgcaagcgca
     4441 attcggctca tcctatggca gccgcgcacg caactcgccc gaactggacg ataaggcccg
     4501 agagcgggca gagcgggcag cactcgccag cttcaaatac aacttcaaaa agcgcctacg
     4561 cgaagtgttg attgtctatc ccgaggcaag cgactgcatc gaagatgacg gcgaatgcct
     4621 gcgcatcaaa tccacacgcc tgcatgtcac ccgcgcaccc ggcaagggcg ctcgcatcgg
     4681 cccccctccg acttgaccag gccaacgcta cgcttggctt ggtcaagcct tcccatccaa
     4741 cagcccgccg tcgagcgggc ttttttatcc ccggaagcct gtggatagag ggtagttatc
     4801 cacgcgaaac cgctaatgcc ccgcaaagcc ttgattcacg gggctttccg gcccgctcca
     4861 aaaactatcc acgtgaaatc gctaatcagg gtacgtgaaa tcgctaatcg gagtacgtga
     4921 aatcgctaat aaggtcacgt gaaatcgcta atcaaaaagg cacgtgagaa cgctaatagc
     4981 cctttcagat caacagcttg caaacacccc tcgctccggc aagtagttac agcaagtagt
     5041 atgttcaatt agcttttcaa ttatgaatat atatatcaat tattggtcgc ccttggcttg
     5101 tggacaatgc gctacgcgca ccggctccgc ccgtggacaa ccgcaagcgg ttgcccaccg
     5161 tcgagcgcct ttgcccacaa cccggcggcc gcaacagatc gttttataaa tttttttttt
     5221 tgaaaaagaa aaagcccgaa aggcggcaac ctctcgggct tctggatttc cgatcaacgc
     5281 aggagtcgtt cggaaagtag ctgttccaga attataggcg cagagacacc agattccaag
     5341 atggctctgt taaattgttg tagtatgtag tatcatacaa catactacag tacagaggcc
     5401 cgcaagaatg gcaatcacta aacaagacat ttggcgagca gccgacgaac tggacgccga
     5461 aggcatccgg cccactttgg ccgccgtgcg caagaaactc ggaagcggta gcttcacaac
     5521 catttccgat gcaatggctg aatggaaaaa ccgcaagacc gccaccctgc cctcatcaga
     5581 cccattgccg gttgcagtca acgagcatct tgccgagctt ggcaatgcgc tatgggctat
     5641 cgccctggcg cacgccaacg cccggtttga cgaagatcgg aaacagatcg aggccgacaa
     5701 agcggccatc agccagcagc ttgccgaagc aatcgaacta gccgacacct tcacccgcga
     5761 aaacgaccag ctccgcgaac gagtgaatca gctcgaacct atggaacgcg agcgcgacaa
     5821 gctggccgac cagcttgccg aagtgaaacg ccgcagcggc gaagaactaa accgctgcat
     5881 ggaaaagctc acccaacgcg ataacgaggc tatcgaggcc cgcaaacagg ccaaggaggc
     5941 catcgagcgc gccgccagtc tgcaaggtca ggtggaagcc ctcaaagagc aggtcgccaa
     6001 tctcacagcc gtcttgaaaa caggaggcaa acaatgaaaa gcgcccttgc cgcccttcgc
     6061 gcggtcgcgg ccgctgtcgt cctaatcgtc agtgtgcccg cttgggccga cttccggggt
     6121 gaagtcgtcc gaatccttga cggtgacact atcgacgttt tggtgaaccg tcagaccatc
     6181 cgcgtgagat tggccgatat tgacgcaccg gaaagcggcc aagccttcgg ctcccgtgct
     6241 cgccaacggc tcgccgactt gacctttcgc caagaggttc aagtgaccga aaaagaggtt
     6301 gatcggtatg gccgcactct tggggtcgtt tacgcgccgt tgcaataccc cggcggccaa
     6361 acacaactca ccaacatcaa tgcgatcatg gttcaagaag gcatggcctg ggcttaccgt
     6421 tattacggca aaccaaccga cgcgcagatg tacgagtatg aaaaagaggc ccgccgccaa
     6481 cggctcggcc tttggtcaga cccgaatgct caggagcctt ggaaatggcg tcgcgcctcg
     6541 aaaaatgcca cgaactgaca ccgggcacgc cccttgttcg acgcgccgca ggcacgtcga
     6601 atttaccgcc gggacgcccc tcgtcccgac acttccagat cgccatagcg cacagcgcct
     6661 cgagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg
     6721 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     6781 tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca
     6841 tcatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca
     6901 tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
     6961 gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg
     7021 aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga
     7081 gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc
     7141 gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag
     7201 gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag
     7261 aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac
     7321 aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
     7381 ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg
     7441 gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt
     7501 atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg
     7561 cctcgcgcac agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg
     7621 tagtcggcaa ataatgtcta acaattcgtt caagccgacg ccgcttcgcg gcgcggctta
     7681 actcaagcgt tagatgcact aagcacataa ttgctcacag ccaaactatc aggtcaagtc
     7741 tgcttttatt atttttaagc gtgcataata agccctacac aaatggtacc ctccggcaaa
     7801 aaaacgggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag attacttcgc
     7861 gtttgccacc tgacgtctaa gaaaaggaat attcagcaat ttgcccgtgc cgaagaaagg
     7921 cccacccgtg aaggtgagcc agtgagttga ttgctacgta attagttagt tagcccttag
     7981 tgactcgaat tcttggcagc ttctagagca attcgcgcta acttacatta attgcgttgc
     8041 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     8101 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     8161 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     8221 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     8281 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     8341 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     8401 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     8461 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     8521 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     8581 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     8641 gagaaaataa tactgttgat gggtgtctgg tcatagacat caagaaataa cgccggaaca
     8701 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     8761 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     8821 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     8881 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     8941 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     9001 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     9061 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     9121 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     9181 gttttgcacc attcgatggt gtcaacgtaa atgcatgccg cttcgccttc gcgcgcgaat
     9241 tggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     9301 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     9361 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     9421 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     9481 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatccgg
     9541 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     9601 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     9661 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     9721 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcgctca caatttacta
     9781 gatgcagttt tattctctcg ccagcactgt aataggcact aatatccaaa ccagtagctc
     9841 aattggagtc gtctat
//
