LOCUS       sequence-254935-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E. coli GreB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  AUTHORS   Tetone LE, Friedman LJ, Osborne ML, Ravi H, Kyzer S, Stumper SK, 
            Mooney RA, Landick R, Gelles J
  TITLE     Dynamics of GreB-RNA polymerase interaction allow a proofreading 
            accessory protein to patrol for transcription complexes needing 
            rescue.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Feb 14;114(7):E1081-E1090. doi: 
            10.1073/pnas.1616525114. Epub 2017 Jan 30.
  PUBMED    28137878
REFERENCE   2  (bases 1 to 6172)
  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.1616525114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-02-14- 14"; volume: "114"; issue: "7"; pages: 
            "E1081-E1090"
COMMENT     Sequence Label: pET15b-GB
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1..23
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..59
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             69..86
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(616..634)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      630..677
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(852..857,858..874,875..880))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(907..925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        993..1097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1098..1166,1167..1958)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1316..1335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2129..2717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2618..2637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2871..2888
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2903..3042
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2968..2987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3128..3150
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3144..3335)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5690..5709
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5729..5806)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6012..6031
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6115..6134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6115..6133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6134..6158
                     /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)."
ORIGIN
        1 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcatcat catcatcaca
       61 gcagcggcct ggtgccgcgc ggcagccata tgaaaacgcc cctggttacc cgggaagggt
      121 atgaaaaact caaacaagag cttaattatc tctggcgtga agaacgcccg gaggtcacaa
      181 aaaaggtgac ctgggccgca agtctgggcg accgcagcga aaatgctgac tatcagtata
      241 ataaaaagcg tctgcgtgaa atcgaccgtc gcgtgcgcta tctcactaaa tgcctggaaa
      301 atctcaaaat cgtcgattac tcccctcagc aggaaggcaa agtctttttt ggcgcgtggg
      361 tggagattga aaacgacgat ggcgtgactc accgtttccg tattgtcggc tacgatgaaa
      421 tttttggccg taaagattac atctctatcg attccccgat ggcccgcgca ttgctgaaaa
      481 aagaagtcgg cgatctggcg gtggtgaata cccctgccgg ggaagcgagc tggtatgtta
      541 atgctatcga gtacgtgaaa ccgtaggatc cggctgctaa caaagcccga aaggaagctg
      601 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      661 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
      721 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
      781 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
      841 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
      901 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
      961 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1021 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1081 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1141 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1201 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1261 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1321 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1381 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1441 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1501 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1561 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1621 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1681 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1741 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     1801 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     1861 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     1921 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     1981 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2041 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2101 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2161 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2221 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2281 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2341 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2401 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2461 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2521 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2581 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2641 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2701 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     2761 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     2821 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     2881 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     2941 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3001 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3061 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3121 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3181 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3241 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3301 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3361 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3421 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     3481 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     3541 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     3601 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     3661 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     3721 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     3781 agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg
     3841 ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca
     3901 gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc
     3961 cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga
     4021 ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg
     4081 aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag
     4141 ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg
     4201 cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca
     4261 tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg
     4321 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4381 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4441 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4501 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4561 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4621 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aactgtcgtg
     4681 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4741 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4801 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4861 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4921 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4981 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5041 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5101 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5161 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5221 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5281 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5341 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5401 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5461 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5521 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5581 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5641 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5701 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5761 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5821 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5881 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5941 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6001 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6061 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     6121 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata at
//
