LOCUS       sequence-106015-        7154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T3-R5-RRVH RNAP mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7154)
  AUTHORS   Meyer AJ, Ellefson JW, Ellington AD
  TITLE     Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase 
            Variants for in Vivo or in Vitro Synthetic Circuitry.
  JOURNAL   ACS Synth Biol. 2014 Oct 7.
  PUBMED    25279711
REFERENCE   2  (bases 1 to 7154)
  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: "ACS Synth 
            Biol. 2014 Oct 7."
COMMENT     Sequence Label: pQE-WT-T3-R5-RRVH
FEATURES             Location/Qualifiers
     source          1..7154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2821..2915
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2959..3618
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3025..3044)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3683..3769
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3862..4944)
                     /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     4906..4925
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4945..5022)
                     /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."
     primer_bind     complement(5106..5128)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5214..5354
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5269..5288
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5369..5386)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5540..6128)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5620..5639)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6235..7050)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6235..7050)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6958..6977
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7042..7061
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     7122..7140
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc aacacgatta acatcgctaa gaacgacttc
      181 tctgacatcg aactggctgc tatcccgttc aacactctgg ctgaccatta cggtgagcgt
      241 ttagctcgcg aacagttggc ccttgagcat gagtcttacg agatgggtga agcacgcttc
      301 cgcaagatgt ttgagcgtca acttaaagct ggtgaggttg cggataacgc tgccgccaag
      361 cctctcatca ctaccctact ccctaagatg attgcacgca tcaacgactg gtttgaggaa
      421 gtgaaagcta agcgcggcaa gcgcccgaca gccttccagt tcctgcaaga aatcaagccg
      481 gaagccgtag cgtacatcac cattaagacc actctggctt gcctaaccag tgctgacaat
      541 acaaccgttc aggctgtagc aagcgcaatc ggtcgggcca ttgaggacga ggctcgcttc
      601 ggtcgtatcc gtgaccttga agctaagcac ttcaagaaaa acgttgagga acaactcaac
      661 aagcgcgtag ggcacgtcta caagaaagca tttatgcaag ttgtcgaggc tgacatgctc
      721 tctaagggtc tactcggtgg cgaggcgtgg tcttcgtggc ataaggaaga ctctattcat
      781 gtaggagtac gctgcatcga gatgctcatt gagtcaaccg gaatggttag cttacaccgc
      841 caaaatgctg gcgtagtagg tcaagactct gagactatcg aactcgcacc tgaatacgct
      901 gaggctatcg caacccgtgc aggtgcgctg gctggcatct ctccgatgtt ccaaccttgc
      961 gtagttcctc ctaagccgtg gactggcatt actggtggtg gctattgggc taacggtcgt
     1021 cgtcctctgg cgctggtgcg tactcacagt aagaaagcac tgatgcgcta cgaagacgtt
     1081 tacatgcctg aggtgtacaa agcgattaac attgcgcaaa acaccgcatg gaaaatcaac
     1141 aagaaagtcc tagcggtcgc caacgtaatc accaagtgga agcattgtcc ggtcgaggac
     1201 atccctgcga ttgagcgtga agaactcccg atgaaaccgg aagacatcga catgaatcct
     1261 gaggctctca ccgcgtggaa acgtgctgcc gctgctgtgt accgcaagga caaggctcgc
     1321 aagtctcgcc gtatcagcct tgagttcatg cttgagcaag ccaataagtt tgctaaccat
     1381 aaggccatct ggttccctta caacatggac tggcgcggtc gtgtttacgc tgtgtcaatg
     1441 ttcaacccgc aaggtaacga tatgaccaaa ggactgctta cgctggcgaa aggtaaacca
     1501 atcggtaagg aaggttacta ctggctgaaa atccacggtg caaactgtgc gggtgtcgat
     1561 aaggttccgt tccctgagcg catcaagttc attgaggaaa accacgagaa catcatggct
     1621 tgcgctaagt ctccactgga gaacacttgg tgggctgagc aagattctcc gttctgcttc
     1681 cttgcattct gctttgagta cgctggggta cagcaccacg gcctgagcta taactgctcc
     1741 cttccgctgg cgtttgacgg gtcttgctct ggcatccagc acttctccgc gatgctccga
     1801 gatgaggtag gtggtcgcgc ggttaacttg cttcctagtg aaaccgttca ggacatctac
     1861 gggattgttg ctaagaaagt caacgagatt ctacaagcag acgcaatcaa tgggaccgat
     1921 aacgaagtag ttaccgtgac cgatgagaac actggtgaaa tctctgagaa agtcaagctg
     1981 ggcactaagg cactggctgg tcaatggctg gcttacggtg ttactcgcag tgtgactaag
     2041 cgttcagtca tgacgctggc ttacgggtcc aaagagttcg gcttccgtca acaagtgctg
     2101 gaagatacca ttcagccagc tattgattcc ggcaagggtc tgatgttcac tcagccgaat
     2161 caggctgctg gatacatggc taagctgatt tgggaatctg tgagcgtgac ggtggtagct
     2221 gcggttgaag caatgaactg gcttaagtct gctgctaagc tgctggctgc tgaggtcaaa
     2281 gataagaaga ctggagagat tcttcgcaag cgttgcgctg tgcattgggt aactcctgat
     2341 ggtttccctg tgtggcagga atacaagaag cctattcaga agcgcttgga tatgattttc
     2401 ctcagacagt tccgcttaca gcctaccatt aacaccaaca aagatagcga gattgatgca
     2461 cgcaaacagg tgtctggtat cgctcctaac tttgtacaca gccatgacgg tagccacctt
     2521 cgtaagactg tagtgtgggc acacgagaag tacggaatcg aatcttttgc actgattcac
     2581 gactccttcg gtaccattcc ggctgacgct gcgaacctgt tcaaagcagt gcgcgaaact
     2641 atggttgaca catatgagtc ttgtgatgta ctggctgatt tctacgacca gttcgctgac
     2701 cagttgcacg agtctcaatt ggacaaaatg ccagcacttc cggctaaagg taacttgaac
     2761 ctccgtgaca tcttagagtc ggacttcgcg ttcgcgtaaa agcttaatta gctgagcttg
     2821 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     2881 tcggttgccg ccgggcgttt tttattggtg agaatccaag ctagcttggc gagattttca
     2941 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     3001 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     3061 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     3121 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt
     3181 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     3241 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     3301 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     3361 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     3421 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     3481 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     3541 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     3601 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     3661 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3721 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     3781 attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa
     3841 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3901 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     3961 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4021 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4081 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4141 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4201 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4261 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4321 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     4381 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     4441 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     4501 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     4561 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     4621 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     4681 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     4741 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     4801 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     4861 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     4921 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     4981 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt
     5041 gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg tgatgacggt
     5101 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     5161 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     5221 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     5281 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     5341 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5401 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5461 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5521 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5581 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5641 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5701 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5761 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5821 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5881 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5941 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6001 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6061 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6121 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6181 cacgttaagg gattttggtc atggcgttaa gggattttgg tcatgaatta attcttagaa
     6241 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     6301 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     6361 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     6421 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     6481 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     6541 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     6601 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     6661 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     6721 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     6781 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6841 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     6901 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     6961 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     7021 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     7081 agacagtttt attgttcatg accatgacat taacctataa aaataggcgt atcacgaggc
     7141 cctttcgtct tcac
//
