LOCUS       sequence-494985-        5086 bp    DNA     circular SYN 10-DEC-2025
DEFINITION  Plasmid for simultaneous expression of the minimum 21 tRNA set.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5086)
  AUTHORS   Miyachi R, Masuda K, Shimizu Y, Ichihashi N
  TITLE     Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA
            array method.
  JOURNAL   Nat Commun. 2025 Aug 26;16(1):7418. doi: 10.1038/s41467-025-62588-y.
  PUBMED    40858540
REFERENCE   2  (bases 1 to 5086)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-08-26"; volume: "16(1):7418. doi"; pages: " 
            10.1038/s41467-025-62588-y"
COMMENT     Sequence Label: pTwist_21tRNAs_split
FEATURES             Location/Qualifiers
     source          1..5086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1191..1209
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1811..1829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2364..2382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2922..2938)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2922..2938)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2935..2957)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(3110..3698)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3190..3209)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3815..3839)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3939..4730,4731..4799))
                     /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     4562..4581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4800..4904)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5003..5025
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5017..5034
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5018..5034
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
       61 gcagcccggg agatctcgat cccgcgaaaa attctaatac gactcactat agcgggaata
      121 gctcagttgg tagagcacga ccttgccaag gtcggggtcg cgagttcgag tctcgtttcc
      181 cgctccaggg gctatagctc agctgggaga gcgcttgcat ggcatgcaag aggtcagcgg
      241 ttcgatcccg cttagctcca ccagcgcccg tagctcagct ggatagagcg ctgccctccg
      301 gaggcagagg tctcaggttc gaatcctgtc gggcgcgcca ggcgcgttaa caaagcggtt
      361 atgtagcgga ttgcaaatcc gtctagtccg gttcgactcc ggaacgcgcc tccatggggt
      421 atcgccaagc ggtaaggcac cggattctga ttccggcatt ccgaggttcg aatcctcgta
      481 ccccagccag ggtcggcatg gcatctccac ctcctcgcgg tccgacctgg gctacttcgg
      541 taggctaagg gagaagagat aacagatact tcggtatctg ttatctgttt tttttcaaca
      601 gatagccgcg ttcgcgcggc tatctgtttt ttttaggcta ggtggaggct cagtgatgcc
      661 gtactggcta aaacctacgg cggtgctgct cgcgcattcg acattctaat acgactcact
      721 atagcgaagg tggcggaatt ggtagacgcg ctagcttcag gtgttagtgt ccttacggac
      781 gtgggggttc aagtcccccc cctcgcacca aggggcgtag ttcaattggt agagcaccgg
      841 tctccaaaac cgggtgttgg gagttcgagt ctctccgccc ctgccaggtg gggttcccga
      901 gcggccaaag ggagcagact gtaaatctgc cgtcacagac ttcgaaggtt cgaatccttc
      961 ccccaccacc agggtcggca tggcatctcc acctcctcgc ggtccgacct gggctacttc
     1021 ggtaggctaa gggagaagag ataacagata cttcggtatc tgttatctgt tttttttcaa
     1081 cagatagccg cgttcgcgcg gctatctgtt ttttttaggc taggtggagg ctcagtgatg
     1141 agatcgataa cgcgccggaa gaaaaagctc gtggtatcac catcaaattc taatacgact
     1201 cactatagga gcggtagttc agtcggttag aatacctgcc tgtcacgcag ggggtcgcgg
     1261 gttcgagtcc cgtccgttcc gccacgcggg gtggagcagc ctggtagctc gtcgggctca
     1321 taacccgaag atcgtcggtt caaatccggc ccccgcaacc aggtggctat agctcagttg
     1381 gtagagccct ggattgtgat tccagttgtc gtgggttcga atcccattag ccaccccagg
     1441 gtcgttagct cagttggtag agcagttgac tcttaatcaa ttggtcgcag gttcgaatcc
     1501 tgcacgaccc accagcgtcc gtagctcagt tggttagagc accaccttga catggtgggg
     1561 gtcggtggtt cgagtccact cggacgcacc agggtcggca tggcatctcc acctcctcgc
     1621 ggtccgacct gggctacttc ggtaggctaa gggagaagag ataacagata cttcggtatc
     1681 tgttatctgt tttttttcaa cagatagccg cgttcgcgcg gctatctgtt ttttttaggc
     1741 taggtggagg ctcagtgatg acacttctca cgttgaatac gacaccccga cccgtcacta
     1801 cgcacaattc taatacgact cactataggt gaggtgtccg agtggctgaa ggagcacgcc
     1861 tggaaagtgt gtatacggca acgtatcggg ggttcgaatc cccccctcac cgccaggcta
     1921 cgtagctcag ttggttagag cacatcactc ataatgatgg ggtcacaggt tcgaatcccg
     1981 tcgtagccac cagcccggat agctcagtcg gtagagcagg ggattgaaaa tccccgtgtc
     2041 cttggttcga ttccgagtcc gggcaccagc tgatatggct cagttggtag agcgcaccct
     2101 tggtaagggt gaggtcccca gttcgactct gggtatcagc accagggtcg gcatggcatc
     2161 tccacctcct cgcggtccga cctgggctac ttcggtaggc taagggagaa gagataacag
     2221 atacttcggt atctgttatc tgtttttttt caacagatag ccgcgttcgc gcggctatct
     2281 gtttttttta ggctaggtgg aggctcagtg atgacgtaga ctgcccgggg cacgccgact
     2341 atgttaaaaa catgatcaaa ttctaatacg actcactata gggcacgtag cgcagcctgg
     2401 tagcgcaccg tcatggggtg tcgggggtcg gaggttcaaa tcctctcgtg ccgaccaagg
     2461 cttgtagctc aggtggttag agcgcacccc tgataagggt gaggtcggtg gttcaagtcc
     2521 actcaggcct accagtcccc ttcgtctaga ggcccaggac accgccctct cacggcggta
     2581 acaggggttc gaatccccta ggggacgcca tcctctgtag ttcagtcggt agaacggcgg
     2641 actgttaatc cgtatgtcac tggttcgagt ccagtcagag gagccagggt cggcatggca
     2701 tctccacctc ctcgcggtcc gacctgggct acttcggtag gctaagggag aagagataac
     2761 agatacttcg gtatctgtta tctgtttttt ttcaacagat agccgcgttc gcgcggctat
     2821 ctgttttttt tgctgaaagg aggaactata tccggataac ctggcaggct aggtggaggc
     2881 tcagtgatga taagtctgcg atggtggatg catgtgtcat ggtcatagct gtttcctgtg
     2941 tgaaattgtt atccgctcag agggcacaat cctattccgc gctatccgac aatctccaag
     3001 acattaggtg gagttcagtt cggcgtatgg catatgtcgc tggaaagaac atgtgagcaa
     3061 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3121 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3181 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3241 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3301 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3361 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3421 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3481 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3541 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3601 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3661 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3721 cggggtctga cgctctattc aacaaagccg ccgtcccgtc aagtcagcgt aaatgggtag
     3781 ggggcttcaa atcgtcctcg tgataccaat tcggagcctg cttttttgta caaacttgtt
     3841 gataatggca attcaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4081 gagccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4141 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4201 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4261 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4321 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4381 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4441 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4501 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4561 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4621 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4681 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4741 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4801 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4861 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4921 gtgccagata cctgaaacaa aacccatcgt acggccaagg aagtctccaa taactgtgat
     4981 ccaccacaag cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtcatgca
     5041 taatccgcac gcatctggaa taaggaagtg ccattccgcc tgacct
//
