LOCUS       sequence-489334-        6106 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses NusG (M1-P124) as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6106)
  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 6106)
  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: pNQ116
FEATURES             Location/Qualifiers
     source          1..6106
                     /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             3171..3188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3241..3259)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3255..3302
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3339..3794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3426..3445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3636..3657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3820..3924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3925..3993,3994..4785)
                     /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(4143..4162)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4956..5544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5445..5464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5698..5715
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5730..5869
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5795..5814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5955..5977
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(5971..6106,1..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 cgtccgtaac
     3121 tagaggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     3181 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     3241 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     3301 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     3361 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3421 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3481 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3541 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3601 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3661 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3721 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3781 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3841 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3901 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3961 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4021 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4081 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4141 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4201 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4261 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4321 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4381 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4441 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     4501 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4561 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4621 tctggagccg gtgagcgtgg ttctcgcggt atcattgcag cactggggcc agatggtaag
     4681 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4741 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4801 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4861 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4921 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4981 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5041 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5101 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5161 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5221 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5281 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5341 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5401 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5461 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5521 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5581 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5641 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5701 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     5761 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5821 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     5881 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5941 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6001 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     6061 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaat
//
