LOCUS       sequence-227327-        6678 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C-terminus of E. coli RfaH with L1 and L2 loops replaced 
            with NusG residues.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6678)
  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 6678)
  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-L1L2NusG
FEATURES             Location/Qualifiers
     source          1..6678
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     620..639
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(659..736)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     942..961
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1045..1064
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1045..1063
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1064..1088
                     /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             1103..1125
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1144..1161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1171..2271
                     /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     2245..2268
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2326..2346
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2587..2604
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2657..2675)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2671..2718
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2755..3210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2842..2861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3052..3073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3303..4118)
                     /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(3303..4118)
                     /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     4026..4045
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4110..4129
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4240..4827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4728..4747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4981..4998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5013..5152
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5078..5097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5238..5260
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5254..5445)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6254..6678,1..658))
                     /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 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
       61 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
      121 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
      181 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
      241 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
      301 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
      361 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
      421 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
      481 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
      541 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
      601 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
      661 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
      721 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
      781 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
      841 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
      901 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
      961 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     1021 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     1081 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac catgggttct
     1141 tctcaccatc accatcacca tggttcttct atgaaaatcg aagaaggtaa actggtaatc
     1201 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
     1261 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
     1321 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
     1381 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
     1441 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
     1501 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg
     1561 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
     1621 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
     1681 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
     1741 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
     1801 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
     1861 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
     1921 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
     1981 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     2041 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     2101 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc
     2161 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     2221 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc
     2281 tcgaacaaca acaacaataa caataacaac aacctcggga tcgaggaaaa cctgtacttc
     2341 caatccaatg cagctactcc ttacccaggc gataaggtaa tcattactga gggtccgttc
     2401 gctgacttcc aagccatttt tacagagcca gacggcgagg ctcgtagcat gttacttctc
     2461 tctatcttcg gtcgtgagat taaacactca gtgaaaaaca ctgagttccg caagttgtaa
     2521 taacattgga agtggataac ggatccgaat tcgagcgccg tcgacaagct tgcggccgca
     2581 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     2641 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2701 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     2761 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2821 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2881 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2941 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3001 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3061 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3121 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3181 attttaacaa actagtaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg
     3241 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     3301 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     3361 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     3421 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     3481 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     3541 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     3601 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3661 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     3721 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tgttttcacc tgaatcagga
     3781 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     3841 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3901 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3961 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     4021 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4081 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4141 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     4201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4261 cgtaatctgc tgcttgcaaa caaaaaacca ccgctaccag cggtggtttg tttgccggat
     4321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4381 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4861 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4921 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4981 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5041 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5101 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5161 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5221 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5281 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5341 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5401 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5461 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5521 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5581 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5641 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5701 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     5761 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5821 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5881 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     5941 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     6001 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     6061 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     6121 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     6181 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     6241 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6301 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     6361 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     6421 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6481 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     6541 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6601 tggcaaccag catcgcagtg ggaacgatac cctcattcag catttgcatg gtttgttgaa
     6661 aaccggacat ggcactcc
//
