LOCUS       sequence-405767-        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pyrrolysyl tRNA variant ""A2.1"" 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 5839)
  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 5839)
  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-PytR(A2.1)-mCherry
FEATURES             Location/Qualifiers
     source          1..5839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   45..174
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        256..504
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     256..276
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     427..446
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        753..1056
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1057..1260
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1211..1231
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1257..1281
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1409..1787
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1865..2575
                     /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(2010..2028)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2278..2297
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2630..3106
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2829..2848)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3146..3286
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3146..3275
                     /label=AAV2 ITR
     rep_origin      3361..3816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3448..3467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3658..3679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3833..3852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3952..3974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4012..4030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4098..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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(4421..4440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5234..5822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5723..5742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
      121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
      181 gcacgcgtct cgcggtccag tagtgatcga cactgctcga tccgctcgca cccctagggg
      241 tacctcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac
      301 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
      361 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      421 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      481 atcttgtgga aaggacgaaa caccggggac ctgatcatgt agatcgaacg gactctaaat
      541 ccgttcagcc gggttagatt cccggggtcc tcgttttttg ctagcggatc gacgagagca
      601 gcgcgactgg atctgtcgcc cgtctcaaac gcaaccctcc ggcggtcgca tatcattcag
      661 gacgagcctc agactccagc gtaacacgcg tctagttatt aatagtaatc aattacgggg
      721 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      781 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      841 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      901 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      961 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1021 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1081 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     1141 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     1201 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     1261 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga
     1321 agacaccggg accgatccag cctccgcgga ttcgaatccc ggccgggaac ggtgcattgg
     1381 aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc tatagagtct ataggcccac
     1441 aaaaaatgct ttcttctttt aatatacttt tttgtttatc ttatttctaa tactttccct
     1501 aatctctttc tttcagggca ataatgatac aatgtatcat gcctctttgc accattctaa
     1561 agaataacag tgataatttc tgggttaagg caatagcaat atttctgcat ataaatattt
     1621 ctgcatataa attgtaactg atgtaagagg tttcatattg ctaatagcag ctacaatcca
     1681 gctaccattc tgcttttatt ttatggttgg gataaggctg gattattctg agtccaagct
     1741 aggccctttt gctaatcatg ttcatacctc ttatcttcct cccacagctc ctgggcaacg
     1801 tgctggtctg tgtgctggcc catcactttg gcaaagaatt gggattcgaa catcgattga
     1861 attcatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     1921 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     1981 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     2041 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     2101 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     2161 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     2221 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     2281 cgtaatgcag aagaagacaa tgggctggga ggcctcctcc gagcggatgt accccgagga
     2341 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     2401 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     2461 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     2521 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaatctag
     2581 agtcgacctg cagaagcttg cctcgagcag cgctgctcga gagatctacg ggtggcatcc
     2641 ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag
     2701 ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
     2761 tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc
     2821 ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat
     2881 ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca
     2941 ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat
     3001 attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa
     3061 attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt
     3121 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     3181 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     3241 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     3301 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     3361 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3421 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3481 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3541 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3601 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3661 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     3721 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     3781 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3841 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3901 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3961 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4021 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5341 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5701 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5761 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5821 aaaacgccag caacgcggc
//
