LOCUS       sequence-485004-        4991 bp    DNA     circular SYN 28-AUG-2025
DEFINITION  In vitro transcription of 21 tRNAs after Nt.BspQI treatment. The 
            plasmid contains a T7 promoter upstream and an Nt.BspQI recognition 
            site downstream of each tRNA gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4991)
  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 4991)
  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: pUC19_21 tRNA genes
FEATURES             Location/Qualifiers
     source          1..4991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        420..438
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        739..757
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        846..864
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        950..968
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1374..1392
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1705..1723
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1811..1829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2131..2149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2461..2479
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2770..2786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2770..2786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2783..2805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2794..2810
                     /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)."
     promoter        complement(join(2818..2824,2825..2842,2843..2848))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2863..2884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3001..3018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3172..3760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3252..3271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3931..4722,4723..4791))
                     /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     4554..4573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4792..4896)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4964..4982
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccccgcgt
      421 aatacgactc actatagggg ctatagctca gctgggagag cgcttgcatg gcatgcaaga
      481 ggtcagcggt tcgatcccgc ttagctccac caagaagagc ccgcgtaata cgactcacta
      541 tagcgcccgt agctcagctg gatagagcgc tgccctccgg aggcagaggt ctcaggttcg
      601 aatcctgtcg ggcgcgccaa gaagagcccg cgtaatacga ctcactatag cctctgtagt
      661 tcagtcggta gaacggcgga ctgttaatcc gtatgtcact ggttcgagtc cagtcagagg
      721 cgccaagaag agcccgcgta atacgactca ctataggagc ggtagttcag tcggttagaa
      781 tacctgcctg tcacgcaggg ggtcgcgggt tcgagtcccg tccgttccgc caagaagagc
      841 ccgcgtaata cgactcacta taggcgcgtt aacaaagcgg ttatgtagcg gattgcaaat
      901 ccgtctagtc cggttcgact ccggaacgcg cctccaagaa gagcccgcgt aatacgactc
      961 actataggcg gggtggagca gcctggtagc tcgtcgggct cataacccga agatcgtcgg
     1021 ttcaaatccg gcccccgcaa ccaagaagag cccgcgtaat acgactcact atatggggta
     1081 tcgccaagcg gtaaggcacc ggattctgat tccggcattc cgaggttcga atcctcgtac
     1141 cccagccaag aagagcccgc gtaatacgac tcactatagt ccccttcgtc tagaggccca
     1201 ggacaccgcc ctctcacggc ggtaacaggg gttcgaatcc cctaggggac gccaagaaga
     1261 gcccgcgtaa tacgactcac tatagcggga atagctcagt tggtagagca cgaccttgcc
     1321 aaggtcgggg tcgcgagttc gagtctcgtt tcccgctcca agaagagccc gcgtaatacg
     1381 actcactata ggtggctata gctcagttgg tagagccctg gattgtgatt ccagttgtcg
     1441 tgggttcgaa tcccattagc caccccaaga agagcccgcg taatacgact cactattagg
     1501 cttgtagctc aggtggttag agcgcacccc tgataagggt gaggtcggtg gttcaagtcc
     1561 actcaggcct accaagaaga gcccgcgtaa tacgactcac tatagcgaag gtggcggaat
     1621 tggtagacgc gctagcttca ggtgttagtg tccttacgga cgtgggggtt caagtccccc
     1681 ccctcgcacc aagaagagcc cgcgtaatac gactcactat agggtcgtta gctcagttgg
     1741 tagagcagtt gactcttaat caattggtcg caggttcgaa tcctgcacga cccaccaaga
     1801 agagcccgcg taatacgact cactataggc tacgtagctc agttggttag agcacatcac
     1861 tcataatgat ggggtcacag gttcgaatcc cgtcgtagcc accaagaaga gcccgcgtaa
     1921 tacgactcac tatagcccgg atagctcagt cggtagagca ggggattgaa aatccccgtg
     1981 tccttggttc gattccgagt ccgggcacca agaagagccc gcgtaatacg actcactata
     2041 cggcacgtag cgcagcctgg tagcgcaccg tcatggggtg tcgggggtcg gaggttcaaa
     2101 tcctctcgtg ccgaccaaga agagcccgcg taatacgact cactataggt gaggtgtccg
     2161 agtggctgaa ggagcacgcc tggaaagtgt gtatacggca acgtatcggg ggttcgaatc
     2221 cccccctcac cgccaagaag agcccgcgta atacgactca ctatagctga tatggctcag
     2281 ttggtagagc gcacccttgg taagggtgag gtccccagtt cgactctggg tatcagcacc
     2341 aagaagagcc cgcgtaatac gactcactat aaggggcgta gttcaattgg tagagcaccg
     2401 gtctccaaaa ccgggtgttg ggagttcgag tctctccgcc cctgccaaga agagcccgcg
     2461 taatacgact cactataggt ggggttcccg agcggccaaa gggagcagac tgtaaatctg
     2521 ccgtcacaga cttcgaaggt tcgaatcctt cccccaccac caagaagagc ccgcgtaata
     2581 cgactcacta tagcgtccgt agctcagttg gttagagcac caccttgaca tggtgggggt
     2641 cggtggttcg agtccactcg gacgcaccaa gaagagcaac cccttggggc ctctaaacgg
     2701 gtcttgaggg gttttttttg gggatcctct agagtcgacc tgcaggcatg caagcttggc
     2761 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2821 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2881 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2941 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3001 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3061 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3121 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3181 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3241 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3301 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3361 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3421 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3481 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3541 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3601 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3661 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3721 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3781 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3841 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3901 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3961 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4021 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4081 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4141 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4201 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4261 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4321 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4381 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4441 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4501 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4561 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4621 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4681 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4741 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4801 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4861 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4921 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4981 gccctttcgt c
//
