LOCUS       sequence-430292-        5852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli tyrosine tRNA variant ""NGS-6"" and a wild-type 
            mCherry reporter; can be packaged into AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5852)
  AUTHORS   Jewel D, Kelemen RE, Huang RL, Zhu Z, Sundaresh B, Cao X, Malley K, 
            Huang Z, Pasha M, Anthony J, van Opijnen T, Chatterjee A
  TITLE     Virus-assisted directed evolution of enhanced suppressor tRNAs in 
            mammalian cells.
  JOURNAL   Nat Methods. 2023 Jan;20(1):95-103. doi: 10.1038/s41592-022-01706-w.
            Epub 2022 Dec 22.
  PUBMED    36550276
REFERENCE   2  (bases 1 to 5852)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-022-01706-w"; journalName: "Nat Methods"; date: 
            "2023-01"; volume: "20"; issue: "1"; pages: "95-103"
COMMENT     Sequence Label: pAAV-ITR-EcYtR(NGS-6)-mCherry
FEATURES             Location/Qualifiers
     source          1..5852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        212..460
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     212..232
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     383..402
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        722..1025
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1026..1229
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1180..1200
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1226..1250
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1378..1756
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1834..2544
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1979..1997)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2247..2266
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2599..3075
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2798..2817)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3115..3255
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3115..3244
                     /label=AAV2 ITR
     rep_origin      3330..3785
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3417..3436)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3627..3648
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3802..3821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3921..3943
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3981..3999)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4067..4171
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4172..4240,4241..5032)
                     /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     complement(4390..4409)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5203..5791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5692..5711
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctcgcg gtccagtagt gatcgacact gctcgatccg
      181 ctcgcacccc taggggtacc tcgggcagga agagggccta tttcccatga ttccttcata
      241 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      301 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      361 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      421 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggggcggttc ccgagcggcc
      481 aaagggagca gactctaaat ctgccgtcac agacttcgaa ggttcgaatc cttcccgccc
      541 cattttttgc tagcggatcg acgagagcag cgcgactgga tctgtcgccc gtctcaaacg
      601 caaccctccg gcggtcgcat atcattcagg acgagcctca gactccagcg taacacgcgt
      661 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      721 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      781 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      841 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      901 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      961 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1021 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1081 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1141 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1201 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
     1261 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggat
     1321 tcgaatcccg gccgggaacg gtgcattgga acgcggattc cccgtgccaa gagtgacgta
     1381 agtaccgcct atagagtcta taggcccaca aaaaatgctt tcttctttta atatactttt
     1441 ttgtttatct tatttctaat actttcccta atctctttct ttcagggcaa taatgataca
     1501 atgtatcatg cctctttgca ccattctaaa gaataacagt gataatttct gggttaaggc
     1561 aatagcaata tttctgcata taaatatttc tgcatataaa ttgtaactga tgtaagaggt
     1621 ttcatattgc taatagcagc tacaatccag ctaccattct gcttttattt tatggttggg
     1681 ataaggctgg attattctga gtccaagcta ggcccttttg ctaatcatgt tcatacctct
     1741 tatcttcctc ccacagctcc tgggcaacgt gctggtctgt gtgctggccc atcactttgg
     1801 caaagaattg ggattcgaac atcgattgaa ttcatggtga gcaagggcga ggaggataac
     1861 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     1921 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     1981 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     2041 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     2101 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     2161 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     2221 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagacaat gggctgggag
     2281 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     2341 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     2401 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     2461 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     2521 ggcatggacg agctgtacaa gtaatctaga gtcgacctgc agaagcttgc ctcgagcagc
     2581 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     2641 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     2701 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     2761 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     2821 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     2881 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     2941 tttttttggt agagacgggg tttcaccata ttggccaggc tcgtctccaa ctcctaatct
     3001 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     3061 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
     3121 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     3181 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     3241 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3301 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3361 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     3421 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3481 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3541 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3601 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3661 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     3721 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3781 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3841 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3901 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3961 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4021 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4081 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4141 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4201 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4261 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4321 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4381 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4441 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4501 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4561 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4621 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4681 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4741 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4801 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4861 tgataaatct ggagccggtg agcgtggctc tcgcggtatc attgcagcac tggggccaga
     4921 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4981 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5041 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5101 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5161 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5221 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5281 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5341 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5401 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5461 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5521 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5581 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5641 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5701 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5761 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5821 cctggccttt tgctggcctt ttgctcacat gt
//
