LOCUS       sequence-223535-        7057 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses full-length E. coli NusG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  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 7057)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: EcNusGFL
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             19..36
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             46..1146
                     /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     1120..1143
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1201..1221
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1840..1857
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1910..1928)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1924..1971
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2008..2463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2095..2114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2305..2326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2556..3371)
                     /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(2556..3371)
                     /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     3279..3298
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3363..3382
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3493..4081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3982..4001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4235..4252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4267..4406
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4332..4351)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4492..4514
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4508..4699)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5508..6590)
                     /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     6552..6571
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6591..6668)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6874..6893
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6977..6996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6977..6995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6996..7020
                     /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             7035..7057
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tataccatgg gttcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa
       61 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
      121 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
      181 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
      241 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
      301 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
      361 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
      421 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
      481 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac
      541 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
      601 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
      661 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
      721 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
      781 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
      841 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
      901 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
      961 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc
     1021 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     1081 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     1141 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag
     1201 gaaaacctgt acttccaatc caatgcaatg tctgaagctc ctaaaaagcg ctggtacgtc
     1261 gttcaggcgt tttccggttt tgaaggccgc gtagcaacgt cgctgcgtga gcatatcaaa
     1321 ttacacaaca tggaagattt gtttggtgaa gtcatggtac caaccgaaga agtggttgaa
     1381 atccgtggcg gtcagcgtcg caaaagcgaa cgtaaattct tccctggcta cgtcctcgtt
     1441 cagatggtga tgaacgacgc gagctggcac ctggtgcgca gcgtaccgcg tgtgatgggc
     1501 ttcatcggcg gtacttccga tcgtcctgcg ccaatcagcg ataaagaagt cgatgcgatt
     1561 atgaaccgcc tgcagcaggt tggtgataag ccgcgtccga aaacgctgtt tgaaccgggt
     1621 gaaatggtcc gtgttaatga tggtccgttc gctgacttca acggtgttgt tgaagaagtg
     1681 gattacgaga aatctcgtct gaaagtgtct gtttctatct tcggtcgtgc gaccccggta
     1741 gagctggact tcagccaggt tgaaaaagcc taataacatt ggaagtggat aacggatccg
     1801 aattcgagcg ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1861 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1921 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1981 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2041 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2101 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2161 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2221 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2281 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2341 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2401 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaactagta acgtttacaa
     2461 tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2521 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2581 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2641 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2701 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2761 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     2821 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2881 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     2941 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3001 agcgcatcaa caatgttttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3061 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3121 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3181 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3241 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3301 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3361 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3421 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3481 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3541 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3601 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     3661 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3721 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3781 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3841 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3901 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3961 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4021 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4081 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4141 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4201 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4261 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4321 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4381 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4441 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4501 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4561 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4621 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     4681 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     4741 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     4801 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     4861 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     4921 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     4981 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5041 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5101 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5161 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     5221 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     5281 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5341 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5401 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5461 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5521 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5581 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     5641 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     5701 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     5761 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     5821 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     5881 ataccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     5941 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     6001 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     6061 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
     6121 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     6181 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     6241 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     6301 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     6361 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
     6421 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     6481 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     6541 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc
     6601 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     6661 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
     6721 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc
     6781 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat
     6841 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
     6901 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
     6961 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     7021 cctctagaaa taattttgtt taactttaag aaggaga
//
