LOCUS       sequence-99358-e        7634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses both fragments of T7 RNAP split at position 179. The 
            N-terminal fragment is driven by PLac while the C-terminal fragment 
            is driven by PBAD. C-terminal fragment has the point mutations 
            R756S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7634)
  AUTHORS   Shis DL, Bennett MR
  TITLE     Library of synthetic transcriptional AND gates built with split T7 
            RNA polymerase mutants.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5028-33. doi: 
            10.1073/pnas.1220157110. Epub 2013 Mar 11.
  PUBMED    23479654
REFERENCE   2  (bases 1 to 7634)
  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"; doi: 
            "10.1073/pnas.1220157110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-03-26- 26"; volume: "110"; issue: "13"; pages: "5028-33"
COMMENT     Sequence Label: pTSlb-R756K
FEATURES             Location/Qualifiers
     source          1..7634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(288..315)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    958..974
                     /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)."
     protein_bind    991..1007
                     /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)."
     primer_bind     1362..1381
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     primer_bind     complement(3677..3694)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3847..3933
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4025..4052
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4071..4162
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4616..5071
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4703..4722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(5626..6285)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6200..6219
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6286..6388)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6780..6797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6914..7459)
                     /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(6994..7013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7531..7550
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
       61 tccgtcaagc cgtcaattgt ctgattcgtt accaacagac attgccgtca ctgcgtcttt
      121 tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa
      181 agcgggacca aagccatgac aaaaacgcgt aatttcaaca cgagccaaaa taataaaaaa
      241 gcaggattaa taatctggct ttttatattc tctctctagt atataaacgc agaaaggccc
      301 acccgaaggt gagccagtgt gatccaagcc agctcgatat ccgtcggctt gaacgaattg
      361 ttagacatta cttgtagacg tgccctacgc gcttgttgag ttgttcctca acgtttttct
      421 tgaagtgctt agcttcaagg tcacggatac gaccgaagcg agcctcgtcc tcaatggccc
      481 gaccgattgc gcttgctaca gcctgaacgg ttgtattgtc agcactggtt aggcaagcca
      541 gagtggtctt aatggtgatg tacgctacgg cttccggctt gatttcttgc aggaactgga
      601 aggctgtcgg gcgcttgccg cgcttagctt tcacttcctc aaaccagtcg ttgatgcgtg
      661 caatcatctt agggagtagg gtagtgatga gaggcttggc ggcagcgtta tccgcaacct
      721 caccagcttt aagttgacgc tcaaacatct tgcggaagcg tgcttcaccc atctcgtaag
      781 actcatgctc aagggccaac tgttcgcgag ctaaacgctc accgtaatgg tcagccagag
      841 tgttgaacgg gatagcagcc agttcgatgt cagagaagtc gttcttagcg atgttaatcg
      901 tgttcatatt agaaaaagaa tgttattaag taagtctttc tagagctctg tgtgaaattg
      961 ttatccgctc acaattgaat ctaagtatca ttgttatccg ctcacaagtc aacactcttt
     1021 ttgataaatt tcctaggact tttctgccgt gattatagac acttttgtcg cgatgttgtg
     1081 tctaattttg aagttaactt tgattccatt cttttgtttg tctgccatga tgtatacatt
     1141 gtgtgagtta tagttgtatt ccaatttgtg tccaagaatg tttccatctt ctttaaaatc
     1201 aatacctttt aactcgattc tattaacaag ggtatcacct tcaaacttga cttcagcacg
     1261 tgtcttgtag ttcccgtcat ctttgaaaaa tatagttctt tcctgtacat aaccttcggg
     1321 cactcgagac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
     1381 cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata
     1441 cccgtttttt tgggctagca ctagtggagc cctatatata aagatcaggg ccaggtatga
     1501 aagcatttat gcaagttgtc gaggctgaca tgctctctaa gggtctactc ggtggcgagg
     1561 cgtggtcttc gtggcataag gaagactcta ttcatgtagg agtacgctgc atcgagatgc
     1621 tcattgagtc aaccggaatg gttagcttac accgccaaaa tgctggcgta gtaggtcaag
     1681 actctgagac tatcgaactc gcacctgaat acgctgaggc tatcgcaacc cgtgcaggtg
     1741 cgctggctgg catctctccg atgttccaac cttgcgtagt tcctcctaag ccgtggactg
     1801 gcattactgg tggtggctat tgggctaacg gtcgtcgtcc tctggcgctg gtgcgtactc
     1861 acagtaagaa agcactgatg cgctacgaag acgtttacat gcctgaggtg tacaaagcga
     1921 ttaacattgc gcaaaacacc gcatggaaaa tcaacaagaa agtcctagcg gtcgccaacg
     1981 taatcaccaa gtggaagcat tgtccggtcg aggacatccc tgcgattgag cgtgaagaac
     2041 tcccgatgaa accggaagac atcgacatga atcctgaggc tctcaccgcg tggaaacgtg
     2101 ctgccgctgc tgtgtaccgc aaggacaagg ctcgcaagtc tcgccgtatc agccttgagt
     2161 tcatgcttga gcaagccaat aagtttgcta accataaggc catctggttc ccttacaaca
     2221 tggactggcg cggtcgtgtt tacgctgtgt caatgttcaa cccgcaaggt aacgatatga
     2281 ccaaaggact gcttacgctg gcgaaaggta aaccaatcgg taaggaaggt tactactggc
     2341 tgaaaatcca cggtgcaaac tgtgcgggtg tcgataaggt tccgttccct gagcgcatca
     2401 agttcattga ggaaaaccac gagaacatca tggcttgcgc taagtctcca ctggagaaca
     2461 cttggtgggc tgagcaagat tctccgttct gcttccttgc gttctgcttt gagtacgctg
     2521 gggtacagca ccacggcctg agctataact gctcccttcc gctggcgttt gacgggtctt
     2581 gctctggcat ccagcacttc tccgcgatgc tccgagatga ggtaggtggt cgcgcggtta
     2641 acttgcttcc tagtgaaacc gttcaggaca tctacgggat tgttgctaag aaagtcaacg
     2701 agattctaca agcagacgca atcaatggga ccgataacga agtagttacc gtgaccgatg
     2761 agaacactgg tgaaatctct gagaaagtca agctgggcac taaggcactg gctggtcaat
     2821 ggctggctta cggtgttact cgcagtgtga ctaagcgttc agtcatgacg ctggcttacg
     2881 ggtccaaaga gttcggcttc cgtcaacaag tgctggaaga taccattcag ccagctattg
     2941 attccggcaa gggtctgatg ttcactcagc cgaatcaggc tgctggatac atggctaagc
     3001 tgatttggga atctgtgagc gtgacggtgg tagctgcggt tgaagcaatg aactggctta
     3061 agtctgctgc taagctgctg gctgctgagg tcaaagataa gaagactgga gagattcttc
     3121 gcaagcgttg cgctgtgcat tgggtaactc ctgatggttt ccctgtgtgg caggaataca
     3181 agaagcctat tcagacgcgc ttgaacctga tgttcctcgg tcagttcaaa ttacagccta
     3241 ccattaacac caacaaagat agcgagattg atgcacacaa acaggagtct ggtatcgctc
     3301 ctaactttgt acacagccaa gacggtagcc accttcgtaa gactgtagtg tgggcacacg
     3361 agaagtacgg aatcgaatct tttgcactga ttcacgactc cttcggtacc attccggctg
     3421 acgctgcgaa cctgttcaaa gcagtgcgcg aaactatggt tgacacatat gagtcttgtg
     3481 atgtactggc tgatttctac gaccagttcg ctgaccagtt gcacgagtct caattggaca
     3541 aaatgccagc acttccggct aaaggtaact tgaacctccg tgacatctta gagtcggact
     3601 tcgcgttcgc gtaatctaga gtcgacctgc aggcatgcaa gcttggctgt tttggcggat
     3661 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     3721 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     3781 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     3841 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3901 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     3961 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     4021 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     4081 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4141 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4201 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4261 tgctgaagat cagttggggc aaactattaa ctggcgaact acttactcta gcttcccggc
     4321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacgcg
     4621 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4681 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4741 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4801 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4861 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4921 ttgttccaaa cttgaacaac actcaaccct atctcgggct attcttttga tttataaggg
     4981 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5041 aattttaaca aaatattaac gtttacaatt taaaaggatc taggtgaaga tcctttttga
     5101 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5161 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5221 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5281 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     5341 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5401 aatcctgtta ccagtcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat
     5461 aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg
     5521 accgggtcga atttgctttc gaatttctgc cattcatccg cttattatca cttattcagg
     5581 cgtagcacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg
     5641 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     5701 gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     5761 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     5821 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     5881 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     5941 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     6001 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     6061 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     6121 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     6181 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     6241 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     6301 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     6361 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     6421 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     6481 aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat
     6541 gatggtgttt ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag
     6601 ctactgacgg ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg
     6661 tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg
     6721 agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc
     6781 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa
     6841 cggggcggag atttcctgga agatgccagg aagatactta acagggaagt gagagggccg
     6901 cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct
     6961 caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg
     7021 gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat
     7081 ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag
     7141 ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat
     7201 cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat
     7261 tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag
     7321 ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag
     7381 aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg
     7441 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tgctcatgag
     7501 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     7561 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tctgctcatg
     7621 tttgacagct tatc
//
