LOCUS       sequence-197937-        7153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector for  expression of single E.coli RNAP subunit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7153)
  AUTHORS   Svetlov V, Artsimovitch I
  TITLE     Purification of bacterial RNA polymerase: tools and protocols.
  JOURNAL   Methods Mol Biol. 2015;1276:13-29. doi: 10.1007/978-1-4939-2392-2_2.
  PUBMED    25665556
REFERENCE   2  (bases 1 to 7153)
  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: "Methods Mol
            Biol."; date: "2015"; volume: "1276:13-29. doi"; pages: " 
            10.1007/978-1-4939-2392-2_2"
COMMENT     Sequence Label: PIA586
FEATURES             Location/Qualifiers
     source          1..7153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     677..696
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(716..793)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     999..1018
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1102..1121
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1102..1120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1121..1145
                     /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             1160..1182
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1201..1218
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1228..1245
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3115..3132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3185..3203)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3199..3246
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3283..3738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3370..3389)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3580..3601
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3831..4646)
                     /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(3831..4646)
                     /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     4554..4573
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4638..4657
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4768..5356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5257..5276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5510..5527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5542..5684
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5610..5629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5770..5792
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5786..5977)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6786..7153,1..715))
                     /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"
ORIGIN
        1 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
       61 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      121 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
      181 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
      241 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
      301 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
      361 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
      421 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      481 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
      541 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
      601 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
      661 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
      721 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
      781 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
      841 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
      901 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
      961 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1021 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     1081 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     1141 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
     1201 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggagcaaaac
     1261 ccgcagtcac agctgaaact tcttgtcacc cgtggtaagg agcaaggcta tctgacctat
     1321 gccgaggtca atgaccatct gccggaagat atcgtcgatt cagatcagat cgaagacatc
     1381 atccaaatga tcaacgacat gggcattcag gtgatggaag aagcaccgga tgccgatgat
     1441 ctgatgctgg ctgaaaacac cgcggacgaa gatgctgccg aagccgccgc gcaggtgctt
     1501 tccagcgtgg aatctgaaat cgggcgcacg actgacccgg tacgcatgta catgcgtgaa
     1561 atgggcaccg ttgaactgtt gacccgcgaa ggcgaaattg acatcgctaa gcgtattgaa
     1621 gacgggatca accaggttca atgctccgtt gctgaatatc cggaagcgat cacctatctg
     1681 ctggaacagt acgatcgtgt tgaagcagaa gaagcgcgtc tgtccgatct gatcaccggc
     1741 tttgttgacc cgaacgcaga agaagatctg gcacctaccg ccactcacgt cggttctgag
     1801 ctttcccagg aagatctgga cgatgacgaa gatgaagacg aagaagatgg cgatgacgac
     1861 agcgccgatg atgacaacag catcgacccg gaactggctc gcgaaaaatt tgcggaacta
     1921 cgcgctcagt acgttgtaac gcgtgacacc atcaaagcga aaggtcgcag tcacgctacc
     1981 gctcaggaag agatcctgaa actgtctgaa gtattcaaac agttccgcct ggtgccgaag
     2041 cagtttgact acctggtcaa cagcatgcgc gtcatgatgg accgcgttcg tacgcaagaa
     2101 cgtctgatca tgaagctctg cgttgagcag tgcaaaatgc cgaagaaaaa cttcattacc
     2161 ctgtttaccg gcaacgaaac cagcgatacc tggttcaacg cggcaattgc gatgaacaag
     2221 ccgtggtcgg aaaaactgca cgatgtctct gaagaagtgc atcgcgccct gcaaaaactg
     2281 cagcagattg aagaagaaac cggcctgacc atcgagcagg ttaaagatat caaccgtcgt
     2341 atgtccatcg gtgaagcgaa agcccgccgt gcgaagaaag agatggttga agcgaactta
     2401 cgtctggtta tttctatcgc taagaaatac accaaccgtg gcttgcagtt ccttgacctg
     2461 attcaggaag gcaacatcgg tctgatgaaa gcggttgata aattcgaata ccgccgtggt
     2521 tacaagttct ccacctacgc aacctggtgg atccgtcagg cgatcacccg ctctatcgcg
     2581 gatcaggcgc gcaccatccg tattccggtg cacatgattg agaccatcaa caagctcaac
     2641 cgtatttctc gccagatgct gcaagagatg ggccgtgaac cgacgccgga agaactggct
     2701 gaacgtatgc tgatgccgga agacaagatc cgcaaagtgc tgaagatcgc caaagagcca
     2761 atctccatgg aaacgccgat cggtgatgat gaagattcgc atctggggga tttcatcgag
     2821 gataccaccc tcgagctgcc gctggattct gcgaccaccg aaagcctgcg tgcggcaacg
     2881 cacgacgtgc tggctggcct gaccgcgcgt gaagcaaaag ttctgcgtat gcgtttcggt
     2941 atcgatatga acaccgacca cacgctggaa gaagtgggta aacagttcga cgttacccgc
     3001 gaacgtatcc gtcagatcga agcgaaggcg ctgcgcaaac tgcgtcaccc gagccgttct
     3061 gaagtgctgc gtagcttcct ggacgattaa agcttgcggc cgcactcgat cgagcaccac
     3121 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     3181 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     3241 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     3301 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3361 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3421 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3481 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3541 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3601 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3661 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3721 tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3781 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     3841 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     3901 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     3961 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4021 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4081 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4141 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4201 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     4261 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4321 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     4381 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     4441 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4501 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4561 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     4621 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     4681 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4741 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4801 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4861 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     4921 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4981 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5041 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5101 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5161 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5221 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5281 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5341 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5401 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5461 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     5521 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5581 ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5641 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     5701 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5761 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5821 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5881 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5941 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     6001 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     6061 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     6121 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     6181 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     6241 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     6301 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     6361 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     6421 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     6481 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     6541 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     6601 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     6661 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     6721 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6781 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6841 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     6901 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     6961 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     7021 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     7081 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     7141 agcatcgcag tgg
//
