LOCUS       sequence-223555-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C-terminus of E. coli RfaH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  AUTHORS   Lawson MR, Ma W, Bellecourt MJ, Artsimovitch I, Martin A, Landick R,
            Schulten K, Berger JM
  TITLE     Mechanism for the Regulated Control of Bacterial Transcription 
            Termination by a Universal Adaptor Protein.
  JOURNAL   Mol Cell. 2018 Sep 20;71(6):911-922.e4. doi: 
            10.1016/j.molcel.2018.07.014. Epub 2018 Aug 16.
  PUBMED    30122535
REFERENCE   2  (bases 1 to 6679)
  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: 
            "10.1016/j.molcel.2018.07.014"; journalName: "Mol Cell"; date: 
            "2018-09-20- 20"; volume: "71"; issue: "6"; pages: "911-922.e4"
COMMENT     Sequence Label: EcRfaHKOW
FEATURES             Location/Qualifiers
     source          1..6679
                     /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..1208
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1182..1205
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1263..1283
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1524..1541
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1594..1612)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1608..1655
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1692..2147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1779..1798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1989..2010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2240..3055)
                     /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(2240..3055)
                     /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     2963..2982
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3047..3066
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3177..3765
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3666..3685
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3919..3936
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3951..4090
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4016..4035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4176..4198
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4192..4383)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5192..6274)
                     /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"
     primer_bind     6236..6255
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6275..6352)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6558..6577
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6661..6679,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6661..6679
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggttcttct caccatcacc atcaccatgg ttcttctatg aaaatcgaag
      121 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
      181 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
      241 aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
      301 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
      361 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
      421 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
      481 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
      541 agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg
      601 acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg
      661 ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca
      721 tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga
      781 tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg
      841 taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg
      901 caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc
      961 tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc
     1021 tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg
     1081 cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc
     1141 gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg
     1201 cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg
     1261 aggaaaacct gtacttccaa tccaatgcag ctactcctta cccaggcgat aaggtaatta
     1321 tcactgaagg agcgtttgaa ggatttcaag ccatttttac agagccagac ggcgaggctc
     1381 gtagcatgtt acttctcaac ctgatcaaca aagagattaa acactcagtg aaaaacactg
     1441 agttccgcaa gttgtaataa cattggaagt ggataacgga tccgaattcg agcgccgtcg
     1501 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     1561 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1621 ttggggcctc taaacgggtc tcgaggggtt ttttgctgaa aggaggaact atatccggat
     1681 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2101 acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag gtggcacttt
     2161 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2221 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     2281 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     2341 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     2401 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     2461 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     2521 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     2581 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     2641 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt
     2701 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     2761 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     2821 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     2881 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     2941 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3001 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     3061 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3121 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3181 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3241 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3301 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3361 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3421 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3481 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3541 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3601 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3661 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3721 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3781 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3841 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3901 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3961 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4021 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4081 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4141 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4201 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4261 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     4321 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     4381 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     4441 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     4501 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     4561 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     4621 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     4681 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     4741 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     4801 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     4861 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     4921 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     4981 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5041 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5101 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5161 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5221 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5281 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     5341 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     5401 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     5461 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     5521 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgataccc tcattcagca
     5581 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     5641 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     5701 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     5761 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     5821 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     5881 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     5941 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6001 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6061 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6121 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     6181 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     6241 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     6301 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     6361 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     6421 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6481 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6541 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6601 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     6661 taatacgact cactatagg
//
