LOCUS       sequence-255639-        6172 bp    DNA     circular SYN 15-APR-2025
DEFINITION  express E. coli GreB E82C/C68S/D41N.
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 Apr 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-GreBdm-D41N
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(519..537)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      533..580
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(755..760,761..777,778..783))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(810..828)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        896..1000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1001..1069,1070..1861)
                     /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(1219..1238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2032..2620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2521..2540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2774..2791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2806..2945
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2871..2890)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3031..3053
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3047..3238)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5593..5612
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5632..5709)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5915..5934
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6018..6037
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6018..6036
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6037..6061
                     /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             6076..6098
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6117..6134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6144..6161
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 gcccctggtt acccgggaag ggtatgaaaa actcaaacaa gagcttaatt atctctggcg
       61 tgaagaacgc ccggaggtca caaaaaaggt gacctgggcc gcaagtctgg gcaaccgcag
      121 cgaaaatgct gactatcagt ataataaaaa gcgtctgcgt gaaatcgacc gtcgcgtgcg
      181 ctatctcact aaaagtctgg aaaatctcaa aatcgtcgat tactcccctc agcagtgcgg
      241 caaagtcttt tttggcgcgt gggtggagat tgaaaacgac gatggcgtga ctcaccgttt
      301 ccgtattgtc ggctacgatg aaatttttgg ccgtaaagat tacatctcta tcgattcccc
      361 gatggcccgc gcattgctga aaaaagaagt cggcgatctg gcggtggtga atacccctgc
      421 cggggaagcg agctggtatg ttaatgctat cgagtacgtg aaaccgtagg atccggctgc
      481 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      541 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      601 cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
      661 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
      721 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
      781 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
      841 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
      901 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
      961 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1021 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1081 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1141 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1201 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     1261 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1321 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1381 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1441 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1501 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     1561 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1621 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     1681 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     1741 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     1801 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     1861 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     1921 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     1981 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2041 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2101 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2161 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2221 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2281 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2341 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2401 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2461 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2521 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2581 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2641 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2701 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2761 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2821 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2881 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     2941 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3001 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3061 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3121 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3181 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3241 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3301 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3361 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3421 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3481 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3541 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3601 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3661 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
     3721 tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc
     3781 aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag
     3841 tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca
     3901 tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc
     3961 caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat
     4021 gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc
     4081 atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag
     4141 aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc
     4201 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4261 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4321 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4381 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4441 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4501 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4561 tttccagtcg ggaaactgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4621 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4681 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4741 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4801 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     4861 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     4921 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     4981 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5041 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5101 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5161 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5221 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5281 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5341 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5401 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5461 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5521 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5581 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5641 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5701 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5761 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     5821 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     5881 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     5941 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6001 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     6061 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     6121 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatgaaa ac
//
