LOCUS       sequence-254934-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express E. coli GreB E82C/C68S.
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-GBdm
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(655..673)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      669..716
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(891..896,897..913,914..919))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(946..964)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1032..1136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1137..1205,1206..1997)
                     /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(1355..1374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2168..2756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2657..2676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2910..2927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2942..3081
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3007..3026)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3167..3189
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3183..3374)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5729..5748
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5768..5845)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6051..6070
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6154..6172,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6154..6172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat gaaaacgccc ctggttaccc gggaagggta tgaaaaactc aaacaagagc
      181 ttaattatct ctggcgtgaa gaacgcccgg aggtcacaaa aaaggtgacc tgggccgcaa
      241 gtctgggcga ccgcagcgaa aatgctgact atcagtataa taaaaagcgt ctgcgtgaaa
      301 tcgaccgtcg cgtgcgctat ctcactaaaa gtctggaaaa tctcaaaatc gtcgattact
      361 cccctcagca gtgcggcaaa gtcttttttg gcgcgtgggt ggagattgaa aacgacgatg
      421 gcgtgactca ccgtttccgt attgtcggct acgatgaaat ttttggccgt aaagattaca
      481 tctctatcga ttccccgatg gcccgcgcat tgctgaaaaa agaagtcggc gatctggcgg
      541 tggtgaatac ccctgccggg gaagcgagct ggtatgttaa tgctatcgag tacgtgaaac
      601 cgtaggatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
      661 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
      721 aaggaggaac tatatccgga tatcccgcaa gaggcccggc agtaccggca taaccaagcc
      781 tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt
      841 catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt
      901 atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc
      961 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     1021 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1081 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1141 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1201 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1261 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1321 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     1381 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1441 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1501 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1561 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1621 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1681 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1741 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1801 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     1861 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1921 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1981 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2041 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2101 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2161 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2221 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2281 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2341 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2401 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2461 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2521 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2581 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2641 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2701 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2761 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2821 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2881 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2941 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3001 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3061 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3121 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3181 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3241 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3301 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3361 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3421 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3481 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3541 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     3601 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3661 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3721 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3781 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3841 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg
     3901 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg
     3961 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
     4021 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     4081 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     4141 cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     4201 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     4261 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg
     4321 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
     4381 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4441 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4501 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4561 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4621 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4681 tgcgctcact gcccgctttc cagtcgggaa actgtcgtgc cagctgcatt aatgaatcgg
     4741 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4801 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4861 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4921 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4981 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5041 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5101 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5161 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5221 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5281 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5341 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5401 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5461 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5521 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5581 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5641 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5701 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5761 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5821 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5881 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5941 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6001 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6061 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6121 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gg
//
