LOCUS       sequence-489325-        7219 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses NusA as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7219)
  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 7219)
  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: pAC005
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(756..1838)
                     /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     1800..1819
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1839..1916)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2122..2141
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2225..2244
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2225..2243
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2244..2268
                     /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             2283..2305
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2318..2335
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2348..2635
                     /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             2636..4120
                     /codon_start=1
                     /gene="nusA"
                     /product="transcription elongation protein (N utilization 
                     substance protein A)"
                     /label=NusA
                     /note="highly soluble in E. coli"
                     /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV
                     RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR
                     ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL
                     REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE
                     IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA
                     QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT
                     VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD
                     EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED
                     LAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA"
     CDS             4175..4192
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4245..4263)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4259..4306
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4343..4798
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4430..4449)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4640..4661
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4824..4928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4929..4997,4998..5789)
                     /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(5147..5166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5960..6548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6449..6468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6702..6719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6734..6873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6799..6818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6959..6981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6975..7166)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
       61 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
      121 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
      181 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
      241 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
      301 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
      361 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
      421 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
      481 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
      541 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
      601 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
      661 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
      721 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      841 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
      901 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
      961 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     1021 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     1081 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     1141 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     1201 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     1261 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     1321 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     1381 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     1441 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     1501 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     1561 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     1621 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     1681 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     1741 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     1801 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     1861 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     1921 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     1981 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     2041 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     2101 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     2161 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     2221 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     2281 attttgttta actttaagaa ggagatatac catgggtcat caccatcatc atcacgggtc
     2341 cctgcaggac tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc
     2401 tgagactcac atcaatttaa aggtgtccga tggatcttca gagatcttct tcaagatcaa
     2461 aaagaccact cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat
     2521 ggactcctta agattcttgt acgacggtat tagaattcaa gctgatcagg cccctgaaga
     2581 tttggacatg gaggataacg atattattga ggctcaccgc gaacagattg gaggtatgaa
     2641 caaagaaatt ttggctgtag ttgaagccgt atccaatgaa aaggcgctac ctcgcgagaa
     2701 gattttcgaa gcattggaaa gcgcgctggc gacagcaaca aagaaaaaat atgaacaaga
     2761 gatcgacgtc cgcgtacaga tcgatcgcaa aagcggtgat tttgacactt tccgtcgctg
     2821 gttagttgtt gatgaagtca cccagccgac caaggaaatc acccttgaag ccgcacgtta
     2881 tgaagatgaa agcctgaacc tgggcgatta cgttgaagat cagattgagt ctgttacctt
     2941 tgaccgtatc actacccaga cggcaaaaca ggttatcgtg cagaaagtgc gtgaagccga
     3001 acgtgcgatg gtggttgatc agttccgtga acacgaaggt gaaatcatca ccggcgtggt
     3061 gaaaaaagta aaccgcgaca acatctctct ggatctgggc aacaacgctg aagccgtgat
     3121 cctgcgcgaa gatatgctgc cgcgtgaaaa cttccgccct ggcgaccgcg ttcgtggcgt
     3181 gctctattcc gttcgcccgg aagcgcgtgg cgcgcaactg ttcgtcactc gttccaagcc
     3241 ggaaatgctg atcgaactgt tccgtattga agtgccagaa atcggcgaag aagtgattga
     3301 aattaaagca gcggctcgcg atccgggttc tcgtgcgaaa atcgcggtga aaaccaacga
     3361 taaacgtatc gatccggtag gtgcttgcgt aggtatgcgt ggcgcgcgtg ttcaggcggt
     3421 gtctactgaa ctgggtggcg agcgtatcga tatcgtcctg tgggatgata acccggcgca
     3481 gttcgtgatt aacgcaatgg caccggcaga cgttgcttct atcgtggtgg atgaagataa
     3541 acacaccatg gatatcgccg ttgaagccgg taacctggcg caggcgattg gccgtaacgg
     3601 tcagaacgtg cgtctggctt cgcagctgag cggttgggaa ctcaacgtga tgaccgttga
     3661 cgacctgcag gctaagcatc aggcggaagc gcacgcagcg atcgacacct tcaccaaata
     3721 tctcgacatc gacgaagact tcgcgactgt tctggtagaa gaaggcttct cgacgctgga
     3781 agaattggcc tatgtgccga tgaaagagct gttggaaatc gaaggccttg atgagccgac
     3841 cgttgaagca ctgcgcgagc gtgctaaaaa tgcactggcc accattgcac aggcccagga
     3901 agaaagcctc ggtgataaca aaccggctga cgatctgctg aaccttgaag gggtagatcg
     3961 tgatttggca ttcaaactgg ccgcccgtgg cgtttgtacg ctggaagatc tcgccgaaca
     4021 gggcattgat gatctggctg atatcgaagg gttgaccgac gaaaaagccg gagcactgat
     4081 tatggctgcc cgtaatattt gctggttcgg tgacgaagcg taactagagg atccgaattc
     4141 gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     4201 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     4261 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     4321 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4381 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     4441 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4501 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4561 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4621 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4681 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4741 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     4801 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4861 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4921 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4981 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5041 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5101 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5161 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5221 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5281 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5341 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5401 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5461 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5521 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5581 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5641 gtggttctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5701 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5761 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5821 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5881 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6001 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6061 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     6121 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6181 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6241 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6301 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6361 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6421 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6481 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6541 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6601 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6661 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6721 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6781 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     6841 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     6901 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     6961 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     7021 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     7081 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     7141 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     7201 ggggtaatga taccgatga
//
