LOCUS       sequence-482092-        4940 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Over-express E. coli Pol III subunit epsilon in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4940)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 4940)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Ec dnaQ-pCDFDuet
FEATURES             Location/Qualifiers
     source          1..4940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(237..1319)
                     /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     1281..1300
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1320..1397)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1443..1461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1462..1486
                     /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)."
     CDS             1542..1559
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1673..1691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1692..1716
                     /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)."
     terminator      2563..2610
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2785..2790,2791..2807,2808..2813))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2840..2858)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             2919..2963
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      3015..3062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3233..4024)
                     /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"
     promoter        complement(4025..4116)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaggatctc aagaagatcc tttgatcttt tctactgaac cgctctagat ttcagtgcaa
       61 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
      121 tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
      181 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
      241 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
      301 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
      361 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
      421 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
      481 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
      541 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
      601 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
      661 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
      721 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
      781 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
      841 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
      901 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
      961 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     1021 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     1081 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     1141 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     1201 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     1261 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     1321 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     1381 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     1441 attaatacga ctcactatag gggaattgtg agcggataac aattcccctg tagaaataat
     1501 tttgtttaac tttaataagg agatatacca tgggcagcag ccatcaccat catcaccaca
     1561 gccaggatcc gaattcgagc tcggcgcgcc tgcaggtcga caagcttgcg gccgcataat
     1621 gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa attaatacga
     1681 ctcactatag gggaattgtg agcggataac aattccccat cttagtatat tagttaagta
     1741 taagaaggag atatacatat gagcactgca attacacgcc agatcgttct cgataccgaa
     1801 accaccggta tgaaccagat tggtgcgcac tatgaaggcc acaagatcat tgagattggt
     1861 gccgttgaag tggtgaaccg tcgcctgacg ggcaataact tccatgttta tctcaaaccc
     1921 gatcggctgg tggatccgga agcctttggc gtacatggta ttgccgatga atttttgctc
     1981 gataagccca cgtttgccga agtagccgat gagttcatgg actatattcg cggcgcggag
     2041 ttggtgatcc ataacgcagc gttcgatatc ggctttatgg actacgagtt ttcgttgctt
     2101 aagcgcgata ttccgaagac caatactttc tgtaaggtca ccgatagcct tgcggtggcg
     2161 aggaaaatgt ttcccggtaa gcgcaacagc ctcgatgcgt tatgtgctcg ctacgaaata
     2221 gataacagta aacgaacgct gcacggggca ttactcgatg cccagatcct tgcggaagtt
     2281 tatctggcga tgaccggtgg tcaaacgtcg atggcttttg cgatggaagg agagacacaa
     2341 cagcaacaag gtgaagcaac aattcagcgc attgtacgtc aggcaagtaa gttacgcgtt
     2401 gtttttgcga cagatgaaga gattgcagct catgaagccc gtctcgatct ggtgcagaag
     2461 aaaggcggaa gttgcctctg gcgagcataa atacctgtag atccggctgc taacaaagcc
     2521 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     2581 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc
     2641 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
     2701 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc
     2761 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag
     2821 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2881 ataatggttt cttagacgtc ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat
     2941 ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca
     3001 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3061 tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac
     3121 cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg
     3181 ggtcatcgtg gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc
     3241 cgactacctt ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc
     3301 gcgaggccaa gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg
     3361 gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga
     3421 ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat
     3481 cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata
     3541 gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc
     3601 tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga
     3661 agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg
     3721 gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa
     3781 tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg
     3841 ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca
     3901 ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc
     3961 gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc
     4021 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4081 gatacatatt tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg
     4141 ctccgggcgt gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg
     4201 tggataagca ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt
     4261 ccataggctc cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt
     4321 gggagccgtg aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa
     4381 gatccccacc gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt
     4441 ttaccggata cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc
     4501 acacactggt atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa
     4561 ctccccgttc agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg
     4621 gaaaagcacg gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat
     4681 ttgtttagct aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa
     4741 ggacagattt ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa
     4801 ctgacttaac cttcgatcaa accacctccc caggtggttt tttcgaaggt taagtcagtt
     4861 ggggaactgc ttaaccttcg atcaaaccac ctccccaggt ggttttttcg tttacagggc
     4921 aaaagattac gcgcagaaaa
//
