LOCUS       sequence-489332-        7405 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses rS1 as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7405)
  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 7405)
  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: pNQ058
FEATURES             Location/Qualifiers
     source          1..7405
                     /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             4470..4487
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4540..4558)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4554..4601
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4638..5093
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4725..4744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4935..4956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5119..5223
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5224..5292,5293..6084)
                     /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(5442..5461)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6255..6843
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6744..6763
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6997..7014
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7029..7168
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7094..7113)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7254..7276
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(7270..7405,1..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 aggtatgact gaatcttttg
     2761 ctcaactctt tgaagagtcc ttaaaagaaa tcgaaacccg cccgggttct atcgttcgtg
     2821 gcgttgttgt tgctatcgac aaagacgtag tactggttga cgctggtctg aaatctgagt
     2881 ccgccatccc ggctgagcag ttcaaaaacg cccagggcga gctggaaatc caggtaggtg
     2941 acgaagttga cgttgctctg gacgcagtag aagacggctt cggtgaaact ctgctgtccc
     3001 gtgagaaagc taaacgtcac gaagcctgga tcacgctgga aaaagcttac gaagatgctg
     3061 aaactgttac cggtgttatc aacggcaaag ttaagggcgg cttcactgtt gagctgaacg
     3121 gtattcgtgc gttcctgcca ggttctctgg tagacgttcg tccggtgcgt gacactctgc
     3181 acctggaagg caaagagctt gaatttaaag taatcaagct ggatcagaag cgcaacaacg
     3241 ttgttgtttc tcgtcgtgcc gttatcgaat ccgaaaacag cgcagagcgc gatcagctgc
     3301 tggaaaacct gcaggaaggc atggaagtta aaggtatcgt taagaacctc actgactacg
     3361 gtgcattcgt tgatctgggc ggcgttgacg gcctgctgca catcactgac atggcctgga
     3421 aacgcgttaa gcatccgagc gaaatcgtca acgtgggcga cgaaatcact gttaaagtgc
     3481 tgaagttcga ccgcgaacgt acccgtgtat ccctgggcct gaaacagctg ggcgaagatc
     3541 cgtgggtagc tatcgctaaa cgttatccgg aaggtaccaa actgactggt cgcgtgacca
     3601 acctgaccga ctacggctgc ttcgttgaaa tcgaagaagg cgttgaaggc ctggtacacg
     3661 tttccgaaat ggactggacc aacaaaaaca tccacccgtc caaagttgtt aacgttggcg
     3721 atgtagtgga agttatggtt ctggatatcg acgaagaacg tcgtcgtatc tccctgggtc
     3781 tgaaacagtg caaagctaac ccgtggcagc agttcgcgga aacccacaac aagggcgacc
     3841 gtgttgaagg taaaatcaag tctatcactg acttcggtat cttcatcggc ttggacggcg
     3901 gcatcgacgg cctggttcac ctgtctgaca tctcctggaa cgttgcaggc gaagaagcag
     3961 ttcgtgaata caaaaaaggc gacgaaatcg ctgcagttgt tctgcaggtt gacgcagaac
     4021 gtgaacgtat ctccctgggc gttaaacagc tcgcagaaga tccgttcaac aactgggttg
     4081 ctctgaacaa gaaaggcgct atcgtaaccg gtaaagtaac tgcagttgac gctaaaggcg
     4141 caaccgtaga actggctgac ggcgttgaag gttacctgcg tgcttctgaa gcatcccgtg
     4201 accgcgttga agacgctacc ctggttctga gcgttggcga cgaagttgaa gctaaattca
     4261 ccggcgttga tcgtaaaaac cgcgcaatca gcctgtctgt tcgtgcgaaa gacgaagctg
     4321 acgagaaaga tgcaatcgca actgttaaca aacaggaaga tgcaaacttc tccaacaacg
     4381 caatggctga agctttcaaa gcagctaaag gcgagtaact agaggatccg aattcgagct
     4441 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     4501 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     4561 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     4621 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4681 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4741 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4801 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4861 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4921 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4981 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5041 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     5101 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5161 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5221 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5281 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5341 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5401 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5461 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5521 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5581 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5641 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5701 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5761 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5821 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5881 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt
     5941 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6001 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6061 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6121 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6181 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6241 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6301 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6361 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     6421 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6481 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6541 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6601 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6661 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6721 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6781 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6841 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6901 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     6961 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7021 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     7081 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     7141 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     7201 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     7261 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     7321 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     7381 gttgagtttc tccagaagcg ttaat
//
