LOCUS       sequence-200376-        7148 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 7148)
  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 7148)
  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..7148
                     /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             3114..3131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3184..3202)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3198..3245
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3282..3737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3369..3388)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3579..3600
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3829..4644)
                     /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(3829..4644)
                     /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     4552..4571
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4636..4655
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4766..5354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5255..5274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5508..5525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5540..5679
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5605..5624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5765..5787
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5781..5972)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6781..7148,1..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 catatgattg 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 acaccgacta cacgctggaa gaagtgggta aacagttcga cgttacccgc
     3001 gaacgtatcc gtcagatcga agcgaaggcg ctgcgcaaac tgcgtcaccc gagccgttct
     3061 gaagtgctgc gtagcttcct ggacgattaa gcttgcggcc gcactcgatc gagcaccacc
     3121 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     3181 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3241 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     3301 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3361 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3421 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3481 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3541 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3601 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3661 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3721 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3781 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     3841 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     3901 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     3961 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     4021 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     4081 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     4141 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     4201 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     4261 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     4321 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     4381 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     4441 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     4501 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     4561 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     4621 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     4681 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4741 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4801 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4861 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4921 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4981 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5041 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5101 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5161 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5221 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5281 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5341 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5401 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5461 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5521 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5581 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     5641 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     5701 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5761 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     5821 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     5881 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     5941 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     6001 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     6061 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     6121 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     6181 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     6241 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     6301 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     6361 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     6421 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     6481 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     6541 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     6601 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     6661 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     6721 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     6781 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6841 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     6901 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     6961 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     7021 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     7081 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     7141 cgcagtgg
//
