LOCUS       sequence-489333-        6277 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses NusG-F165A as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6277)
  AUTHORS   Qureshi NS, Duss O
  TITLE     Tracking transcription-translation coupling in real time.
  JOURNAL   Nature. 2025 Jan;637(8045):487-495. doi: 10.1038/s41586-024-08308-w.
            Epub 2024 Dec 4.
  PUBMED    39633055
REFERENCE   2  (bases 1 to 6277)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-024-08308-w"; journalName: "Nature"; date: 
            "2025-01"; volume: "637"; issue: "8045"; pages: "487-495"
COMMENT     Sequence Label: pNQ117
FEATURES             Location/Qualifiers
     source          1..6277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(865..1947)
                     /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     1909..1928
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1948..2025)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2231..2250
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2334..2353
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2334..2352
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2353..2377
                     /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             2392..2414
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2427..2444
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2457..2744
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             3342..3359
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3412..3430)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3426..3473
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3510..3965
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3597..3616)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3807..3828
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3991..4095
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4096..4164,4165..4956)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4314..4333)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5127..5715
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5616..5635
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5869..5886
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5901..6040
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5966..5985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6126..6148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6142..6277,1..56))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
       61 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
      121 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
      181 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
      241 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
      301 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
      361 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
      421 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
      481 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
      541 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
      601 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
      661 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
      721 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
      781 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
      841 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      901 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
      961 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     1021 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     1081 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     1141 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     1201 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     1261 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     1321 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     1381 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     1441 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     1501 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     1561 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     1621 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     1681 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     1741 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     1801 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     1861 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     1921 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     1981 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     2041 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     2101 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     2161 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     2221 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     2281 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     2341 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
     2401 ctttaagaag gagatatacc atgggtcatc accatcatca tcacgggtcc ctgcaggact
     2461 cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct gagactcaca
     2521 tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa aagaccactc
     2581 ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg gactccttaa
     2641 gattcttgta cgacggtatt agaattcaag ctgatcaggc ccctgaagat ttggacatgg
     2701 aggataacga tattattgag gctcaccgcg aacagattgg aggtatgtct gaagctccta
     2761 aaaagcgctg gtacgtcgtt caggcgtttt ccggttttga aggccgcgta gcaacgtcgc
     2821 tgcgtgagca tatcaaatta cacaacatgg aagatttgtt tggtgaagtc atggtaccaa
     2881 ccgaagaagt ggttgaaatc cgtggcggtc agcgtcgcaa aagcgaacgt aaattcttcc
     2941 ctggctacgt cctcgttcag atggtgatga acgacgcgag ctggcacctg gtgcgcagcg
     3001 taccgcgtgt gatgggcttc atcggcggta cttccgatcg tcctgcgcca atcagcgata
     3061 aagaagtcga tgcgattatg aaccgcctgc agcaggttgg tgataagccg cgtccgaaaa
     3121 cgctgtttga accgggtgaa atggtccgtg ttaatgatgg tccgttcgct gacttcaacg
     3181 gtgttgttga agaagtggat tacgagaaat ctcgtctgaa agtgtctgtt tctatcgcgg
     3241 gtcgtgcgac cccggtagag ctggacttca gccaggttga aaaagcctaa ctagaggatc
     3301 cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     3361 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     3421 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     3481 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     3541 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3601 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3661 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3721 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3781 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3841 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3901 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3961 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4021 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4081 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4141 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4201 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4261 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4321 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4381 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4441 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4501 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4561 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4621 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     4681 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     4741 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     4801 ggtgagcgtg gttctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4861 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4921 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4981 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5041 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5101 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5161 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5221 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     5281 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5341 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5401 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5461 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5521 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5581 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5641 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5701 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5761 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5821 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5881 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5941 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6001 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     6061 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     6181 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     6241 cgcgtccagc tcgttgagtt tctccagaag cgttaat
//
