LOCUS       sequence-405769-        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pyrrolysyl tRNA 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-mCherry
FEATURES             Location/Qualifiers
     source          1..5839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   90..219
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        301..549
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     301..321
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     472..491
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        798..1101
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1102..1305
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1256..1276
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1302..1326
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1454..1832
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1910..2620
                     /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(2055..2073)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2323..2342
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2675..3151
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2874..2893)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3191..3331
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3191..3320
                     /label=AAV2 ITR
     rep_origin      3406..3861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3493..3512)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3703..3724
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3878..3897)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3997..4019
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4057..4075)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4143..4247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4248..4316,4317..5108)
                     /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(4466..4485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5279..5839,1..28)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5768..5787
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
       61 ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac
      121 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
      181 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtctcgcgg
      241 tccagtagtg atcgacactg ctcgatccgc tcgcacccct aggggtacct cgggcaggaa
      301 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      361 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      421 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      481 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      541 cgaaacaccg gaaacctgat catgtagatc gaacggactc taaatccgtt cagccgggtt
      601 agattcccgg ggtttccgtt ttttgctagc ggatcgacga gagcagcgcg actggatctg
      661 tcgcccgtct caaacgcaac cctccggcgg tcgcatatca ttcaggacga gcctcagact
      721 ccagcgtaac acgcgtctag ttattaatag taatcaatta cggggtcatt agttcatagc
      781 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      841 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      901 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      961 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1021 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1081 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     1141 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1201 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     1261 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
     1321 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
     1381 tccagcctcc gcggattcga atcccggccg ggaacggtgc attggaacgc ggattccccg
     1441 tgccaagagt gacgtaagta ccgcctatag agtctatagg cccacaaaaa atgctttctt
     1501 cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca
     1561 gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata
     1621 atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt
     1681 aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt
     1741 ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa
     1801 tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg gtctgtgtgc
     1861 tggcccatca ctttggcaaa gaattgggat tcgaacatcg attgaattca tggtgagcaa
     1921 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     1981 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     2041 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     2101 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     2161 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     2221 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     2281 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     2341 gacaatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     2401 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     2461 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     2521 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     2581 ccgccactcc accggcggca tggacgagct gtacaagtaa tctagagtcg acctgcagaa
     2641 gcttgcctcg agcagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc
     2701 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
     2761 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
     2821 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     2881 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     2941 tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag
     3001 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     3061 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     3121 ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc
     3181 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3241 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3301 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     3361 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     3421 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3481 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3601 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3721 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     3781 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3841 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     3901 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3961 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4021 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4081 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4141 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4201 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4261 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4321 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4381 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4441 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4501 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4561 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4621 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4681 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4741 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4801 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4861 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4921 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4981 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5041 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5101 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5161 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5221 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5281 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5341 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5401 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5461 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5521 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5581 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5641 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5701 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5761 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5821 agcgtcgatt tttgtgatg
//
