LOCUS       sequence-485003-        7038 bp    DNA     circular SYN 28-AUG-2025
DEFINITION  Expresses 21 tRNAs. The plasmid contains a T7 promoter upstream and 
            an Nt.BspQI recognition site as well as a T7 terminator downstream 
            of each tRNA gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7038)
  AUTHORS   Li F, Baranwal AK, Maerkl SJ
  TITLE     Continuous in situ synthesis of a complete set of tRNAs sustains 
            steady-state translation in a recombinant cell-free system.
  JOURNAL   Nat Commun. 2025 Jul 5;16(1):6212. doi: 10.1038/s41467-025-61671-8.
  PUBMED    40617857
REFERENCE   2  (bases 1 to 7038)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-07-5"; volume: "16(1):6212. doi"; pages: " 
            10.1038/s41467-025-61671-8"
COMMENT     Sequence Label: pETA_21 tRNA genes
FEATURES             Location/Qualifiers
     source          1..7038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             809..1000
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(994..1016)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1102..1244
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1157..1176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1259..1276)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1430..2018)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1510..1529)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2189..2980,2981..3049))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2812..2831
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3050..3154)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3161..3208
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3209..3295
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3387..3414
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3481..3503
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3495..3512
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3496..3512
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3518..3536
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3963..3981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4112..4130
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4258..4276
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4850..4868
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5307..5325
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5455..5473
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5901..5919
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        6357..6375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6670..6686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6670..6686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6683..6705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6694..6710
                     /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)."
     terminator      complement(6755..6782)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6874..6960)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(6961..6966,6967..6984,6985..6991)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             7006..7028
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
       61 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
      121 cgcacttatg actgtgttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
      181 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
      241 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
      301 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
      361 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
      421 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
      481 aacaacatga atggtctttg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
      541 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
      601 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
      661 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
      721 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
      781 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
      841 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
      901 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
      961 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggtgac
     1021 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     1081 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     1141 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt
     1201 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     1261 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1321 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1381 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1441 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1501 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1561 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1621 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1681 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1741 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1801 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1861 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1921 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1981 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2041 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2101 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2161 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2221 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2281 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gayccacgct
     2341 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2401 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2461 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2521 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2581 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2641 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2701 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2761 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2821 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2881 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2941 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3001 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3061 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3121 gtatttagaa aaataaacaa ataggggttc cgcgcctagg ctagcataac cccttggggc
     3181 ctctaaacgg gtcttgaggg gttttttgca aataaaacga aaggctcagt cgaaagactg
     3241 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     3301 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     3361 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     3421 tctacaaact ctctggccct agagaaccca ctgcttacag ttgggtaacg ccagggtttt
     3481 cccagtcacg acgttgtaaa acgacggcca gtccgcgtaa tacgactcac tataggggct
     3541 atagctcagc tgggagagcg cttgcatggc atgcaagagg tcagcggttc gatcccgctt
     3601 agctccacca agaagagcaa ccccttgggg cctctaaacg ggtcttgagg ggtttttttt
     3661 ccgcgtaata cgactcacta tagcgcccgt agctcagctg gatagagcgc tgccctccgg
     3721 aggcagaggt ctcaggttcg aatcctgtcg ggcgcgccaa gaagagcaac cccttggggc
     3781 ctctaaacgg gtcttgaggg gttttttttc cgcgtaatac gactcactat agcctctgta
     3841 gttcagtcgg tagaacggcg gactgttaat ccgtatgtca ctggttcgag tccagtcaga
     3901 ggcgccaaga agagcaaccc cttggggcct ctaaacgggt cttgaggggt tttttttccg
     3961 cgtaatacga ctcactatag gagcggtagt tcagtcggtt agaatacctg cctgtcacgc
     4021 agggggtcgc gggttcgagt cccgtccgtt ccgccaagaa gagcaacccc ttggggcctc
     4081 taaacgggtc ttgaggggtt ttttttccgc gtaatacgac tcactatagg cgcgttaaca
     4141 aagcggttat gtagcggatt gcaaatccgt ctagtccggt tcgactccgg aacgcgcctc
     4201 caagaagagc aaccccttgg ggcctctaaa cgggtcttga ggggtttttt ttccgcgtaa
     4261 tacgactcac tataggcggg gtggagcagc ctggtagctc gtcgggctca taacccgaag
     4321 atcgtcggtt caaatccggc ccccgcaacc aagaagagca accccttggg gcctctaaac
     4381 gggtcttgag gggttttttt tccgcgtaat acgactcact atatggggta tcgccaagcg
     4441 gtaaggcacc ggattctgat tccggcattc cgaggttcga atcctcgtac cccagccaag
     4501 aagagcaacc ccttggggcc tctaaacggg tcttgagggg ttttttttcc gcgtaatacg
     4561 actcactata gtccccttcg tctagaggcc caggacaccg ccctctcacg gcggtaacag
     4621 gggttcgaat cccctagggg acgccaagaa gagcaacccc ttggggcctc taaacgggtc
     4681 ttgaggggtt ttttttccgc gtaatacgac tcactatagc gggaatagct cagttggtag
     4741 agcacgacct tgccaaggtc ggggtcgcga gttcgagtct cgtttcccgc tccaagaaga
     4801 gcaacccctt ggggcctcta aacgggtctt gaggggtttt ttttccgcgt aatacgactc
     4861 actataggtg gctatagctc agttggtaga gccctggatt gtgattccag ttgtcgtggg
     4921 ttcgaatccc attagccacc ccaagaagag caaccccttg gggcctctaa acgggtcttg
     4981 aggggttttt tttccgcgta atacgactca ctattaggct tgtagctcag gtggttagag
     5041 cgcacccctg ataagggtga ggtcggtggt tcaagtccac tcaggcctac caagaagagc
     5101 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt ttccgcgtaa tacgactcac
     5161 tatagcgaag gtggcggaat tggtagacgc gctagcttca ggtgttagtg tccttacgga
     5221 cgtgggggtt caagtccccc ccctcgcacc aagaagagca accccttggg gcctctaaac
     5281 gggtcttgag gggttttttt tccgcgtaat acgactcact atagggtcgt tagctcagtt
     5341 ggtagagcag ttgactctta atcaattggt cgcaggttcg aatcctgcac gacccaccaa
     5401 gaagagcaac cccttggggc ctctaaacgg gtcttgaggg gttttttttc cgcgtaatac
     5461 gactcactat aggctacgta gctcagttgg ttagagcaca tcactcataa tgatggggtc
     5521 acaggttcga atcccgtcgt agccaccaag aagagcaacc ccttggggcc tctaaacggg
     5581 tcttgagggg ttttttttcc gcgtaatacg actcactata gcccggatag ctcagtcggt
     5641 agagcagggg attgaaaatc cccgtgtcct tggttcgatt ccgagtccgg gcaccaagaa
     5701 gagcaacccc ttggggcctc taaacgggtc ttgaggggtt ttttttccgc gtaatacgac
     5761 tcactatacg gcacgtagcg cagcctggta gcgcaccgtc atggggtgtc gggggtcgga
     5821 ggttcaaatc ctctcgtgcc gaccaagaag agcaacccct tggggcctct aaacgggtct
     5881 tgaggggttt tttttccgcg taatacgact cactataggt gaggtgtccg agtggctgaa
     5941 ggagcacgcc tggaaagtgt gtatacggca acgtatcggg ggttcgaatc cccccctcac
     6001 cgccaagaag agcaacccct tggggcctct aaacgggtct tgaggggttt tttttccgcg
     6061 taatacgact cactatagct gatatggctc agttggtaga gcgcaccctt ggtaagggtg
     6121 aggtccccag ttcgactctg ggtatcagca ccaagaagag caaccccttg gggcctctaa
     6181 acgggtcttg aggggttttt tttccgcgta atacgactca ctataagggg cgtagttcaa
     6241 ttggtagagc accggtctcc aaaaccgggt gttgggagtt cgagtctctc cgcccctgcc
     6301 aagaagagca accccttggg gcctctaaac gggtcttgag gggttttttt tccgcgtaat
     6361 acgactcact ataggtgggg ttcccgagcg gccaaaggga gcagactgta aatctgccgt
     6421 cacagacttc gaaggttcga atccttcccc caccaccaag aagagcaacc ccttggggcc
     6481 tctaaacggg tcttgagggg ttttttttcc gcgtaatacg actcactata gcgtccgtag
     6541 ctcagttggt tagagcacca ccttgacatg gtgggggtcg gtggttcgag tccactcgga
     6601 cgcaccaaga agagcaaccc cttggggcct ctaaacgggt cttgaggggt tttttttggc
     6661 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     6721 gaaggtgcca ctcccactgt tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     6781 ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     6841 ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     6901 cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg
     6961 tttacacttt atgcttccgg ctcgtatgtt gcctctagaa ataattttgt ttaactttaa
     7021 gaaggagata tacatatg
//
