LOCUS       sequence-489327-        6676 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses MTF as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6676)
  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 6676)
  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: pNQ056
FEATURES             Location/Qualifiers
     source          1..6676
                     /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             3741..3758
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3811..3829)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3825..3872
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3909..4364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3996..4015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4206..4227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4390..4494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4495..4563,4564..5355)
                     /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(4713..4732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5526..6114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6015..6034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6268..6285
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6300..6439
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6365..6384)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6525..6547
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6541..6676,1..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 aggttcagaa tcactacgta
     2761 ttatttttgc gggtacacct gactttgcag cgcgtcatct cgacgcgctg ttgtcttctg
     2821 gtcataacgt cgttggcgtg ttcacccagc cagaccgacc ggcaggacgc ggtaaaaaac
     2881 tgatgcccag cccggttaaa gttctggctg aggaaaaagg tctgcccgtt tttcaacctg
     2941 tttccctgcg tccacaagaa aaccagcaac tggtcgccga actgcaggct gatgttatgg
     3001 tcgtcgtcgc ctatggttta attctgccga aagcagtgct ggagatgccg cgtcttggct
     3061 gtatcaacgt tcatggttca ctgctgccac gctggcgcgg tgctgcacca atccaacgct
     3121 cactatgggc gggtgatgca gaaactggtg tgaccattat gcaaatggat gtcggtttag
     3181 acaccggtga tatgctctat aagctctcct gcccgattac tgcagaagat accagtggta
     3241 cgctgtacga caagctggca gagcttggcc cacaagggct tatcaccacg ttgaaacaac
     3301 tggcagacgg cacggcgaaa ccagaagttc aggacgaaac tcttgtcact tacgccgaga
     3361 agttgagtaa agaagaagcg cgtattgact ggtcactttc ggcagcacag cttgaacgct
     3421 gcattcgcgc tttcaatcca tggccaatga gctggctgga aattgaagga cagccggtta
     3481 aagtctggaa agcatcggtc attgatacgg caaccaacgc tgcaccagga acgatccttg
     3541 aagccaacaa acaaggcatt caggttgcga ctggtgatgg catcctgaac ctgctctcgt
     3601 tacaacctgc gggtaagaaa gcgatgagcg cgcaagacct cctgaactct cgtcgggaat
     3661 ggtttgttcc gggcaaccgt ctggtctaac tagaggatcc gaattcgagc tccgtcgaca
     3721 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     3781 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     3841 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     3901 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3961 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4021 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4081 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4141 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4201 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4261 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4321 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     4381 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4441 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4501 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4561 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4621 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4681 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4741 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4801 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4861 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4921 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     4981 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     5041 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5101 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5161 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttctcgcggt
     5221 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5281 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5341 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5401 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5641 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6181 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     6241 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     6301 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     6361 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     6421 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     6481 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6541 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     6601 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     6661 ctccagaagc gttaat
//
