LOCUS       sequence-489331-        7378 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Expresses MetRS (truncated at K548) as fusion with 6xHis-SUMO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7378)
  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 7378)
  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: pNQ055
FEATURES             Location/Qualifiers
     source          1..7378
                     /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             4443..4460
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4513..4531)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4527..4574
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4611..5066
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4698..4717)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4908..4929
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5092..5196
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5197..5265,5266..6057)
                     /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(5415..5434)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6228..6816
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6717..6736
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6970..6987
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7002..7141
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7067..7086)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7227..7249
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(7243..7378,1..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 tgtgcactgc
     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 caagtcgcga
     2761 agaaaattct ggtgacgtgc gcactgccgt acgctaacgg ctcaatccac ctcggccata
     2821 tgctggagca catccaggct gatgtctggg tccgttacca gcgaatgcgc ggccacgagg
     2881 tcaacttcat ctgcgccgac gatgcccacg gtacaccgat catgctgaaa gctcagcagc
     2941 ttggtatcac cccggagcag atgattggcg aaatgagtca ggagcatcag actgatttcg
     3001 caggctttaa catcagctat gacaactatc actcgacgca cagcgaagag aaccgccagt
     3061 tgtcagaact tatctactct cgcctgaaag aaaacggttt tattaaaaac cgcaccatct
     3121 ctcagctgta cgatccggaa aaaggcatgt tcctgccgga ccgttttgtg aaaggcacct
     3181 gcccgaaatg taaatccccg gatcaatacg gcgataactg cgaagtctgc ggcgcgacct
     3241 acagcccgac tgaactgatc gagccgaaat cggtggtttc tggcgctacg ccggtaatgc
     3301 gtgattctga acacttcttc tttgatctgc cctctttcag cgaaatgttg caggcatgga
     3361 cccgcagcgg tgcgttgcag gagcaggtgg caaataaaat gcaggagtgg tttgaatctg
     3421 gcctgcaaca gtgggatatc tcccgcgacg ccccttactt cggttttgaa attccgaacg
     3481 cgccgggcaa atatttctac gtctggctgg acgcaccgat tggctacatg ggttctttca
     3541 agaatctgtg cgacaagcgc ggcgacagcg taagcttcga tgaatactgg aagaaagact
     3601 ccaccgccga gctgtaccac ttcatcggta aagatattgt ttacttccac agcctgttct
     3661 ggcctgccat gctggaaggc agcaacttcc gcaagccgtc caacctgttt gttcatggct
     3721 atgtgacggt gaacggcgca aagatgtcca agtctcgcgg cacctttatt aaagccagca
     3781 cctggctgaa tcattttgac gcagacagcc tgcgttacta ctacactgcg aaactctctt
     3841 cgcgcattga tgatatcgat ctcaacctgg aagatttcgt tcagcgtgtg aatgccgata
     3901 tcgttaacaa agtggttaac ctggcctccc gtaatgcggg ctttatcaac aagcgttttg
     3961 acggcgtgct ggcaagcgaa ctggctgacc cgcagttgta caaaaccttc actgatgccg
     4021 ctgaagtgat tggtgaagcg tgggaaagcc gtgaatttgg taaagccgtg cgcgaaatca
     4081 tggcgctggc tgatctggct aaccgctatg tcgatgaaca ggctccgtgg gtggtggcga
     4141 aacaggaagg ccgcgatgcc gacctgcagg caatttgctc aatgggcatc aacctgttcc
     4201 gcgtgctgat gacttacctg aagccggtac tgccgaaact gaccgagcgt gcagaagcat
     4261 tcctcaatac ggaactgacc tgggatggta tccagcaacc gctgctgggc cacaaagtga
     4321 atccgttcaa ggcgctgtat aaccgcatcg atatgaggca ggttgaagca ctggtggaag
     4381 cctctaaata actagaggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg
     4441 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     4501 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     4561 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     4621 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4681 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4741 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4801 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4861 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4921 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4981 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5041 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5101 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5161 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5221 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5281 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5341 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5401 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5461 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5521 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5581 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5641 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5701 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     5761 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5821 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5881 attgctgata aatctggagc cggtgagcgt ggttctcgcg gtatcattgc agcactgggg
     5941 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6001 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6061 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6121 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6181 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6241 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6301 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6361 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6421 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6481 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6541 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6601 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6661 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6721 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6781 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6841 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6901 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6961 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     7021 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     7081 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     7141 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     7201 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     7261 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     7321 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaat
//
