LOCUS       sequence-405770-        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses pyrrolysyl tRNA variant ""PyOtR"" 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, Malley K, Pham Q,
            Loynd C, Huang Z, van Opijnen T, Chatterjee A
  TITLE     Enhanced Directed Evolution in Mammalian Cells Yields a 
            Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid 
            Mutagenesis.
  JOURNAL   Angew Chem Int Ed Engl. 2024 Jan 26:e202316428. doi: 
            10.1002/anie.202316428.
  PUBMED    38279536
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"; journalName: "Angew Chem 
            Int Ed Engl. 2024 Jan 26:e202316428. doi: 10.1002/anie.202316428."
COMMENT     Sequence Label: pAAV-ITR-PytR(PyOtR)-mCherry
FEATURES             Location/Qualifiers
     source          1..5839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   3..132
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        214..462
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     214..234
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     385..404
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        711..1014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1015..1218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1169..1189
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1215..1239
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1367..1745
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1823..2533
                     /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(1968..1986)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2236..2255
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2588..3064
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2787..2806)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3104..3244
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3104..3233
                     /label=AAV2 ITR
     rep_origin      3319..3774
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3406..3425)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3616..3637
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3791..3810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3910..3932
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3970..3988)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4056..4160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4161..4229,4230..5021)
                     /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(4379..4398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5192..5780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5681..5700
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct
       61 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
      121 ctaggggttc ctgcggccgc acgcgtctcg cggtccagta gtgatcgaca ctgctcgatc
      181 cgctcgcacc cctaggggta cctcgggcag gaagagggcc tatttcccat gattccttca
      241 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
      301 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
      361 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
      421 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgggcggct gatcatgtag
      481 atcgaacgga ctctaaatcc gttcagccgg gttagattcc cgggctgccc gttttttgct
      541 agcggatcga cgagagcagc gcgactggat ctgtcgcccg tctcaaacgc aaccctccgg
      601 cggtcgcata tcattcagga cgagcctcag actccagcgt aacacgcgtc tagttattaa
      661 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
      721 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      781 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
      841 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
      901 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
      961 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     1021 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     1081 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     1141 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     1201 gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
     1261 tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggatt cgaatcccgg
     1321 ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta
     1381 tagagtctat aggcccacaa aaaatgcttt cttcttttaa tatacttttt tgtttatctt
     1441 atttctaata ctttccctaa tctctttctt tcagggcaat aatgatacaa tgtatcatgc
     1501 ctctttgcac cattctaaag aataacagtg ataatttctg ggttaaggca atagcaatat
     1561 ttctgcatat aaatatttct gcatataaat tgtaactgat gtaagaggtt tcatattgct
     1621 aatagcagct acaatccagc taccattctg cttttatttt atggttggga taaggctgga
     1681 ttattctgag tccaagctag gcccttttgc taatcatgtt catacctctt atcttcctcc
     1741 cacagctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattgg
     1801 gattcgaaca tcgattgaat tcatggtgag caagggcgag gaggataaca tggccatcat
     1861 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     1921 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     1981 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     2041 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     2101 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     2161 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     2221 cttcccctcc gacggccccg taatgcagaa gaagacaatg ggctgggagg cctcctccga
     2281 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     2341 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     2401 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     2461 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     2521 gctgtacaag taatctagag tcgacctgca gaagcttgcc tcgagcagcg ctgctcgaga
     2581 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     2641 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     2701 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     2761 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     2821 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     2881 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     2941 gagacggggt ttcaccatat tggccaggct cgtctccaac tcctaatctc aggtgatcta
     3001 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     3061 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     3121 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     3181 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     3241 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     3301 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3361 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     3421 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3481 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     3541 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3601 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     3661 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     3721 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     3781 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3841 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3901 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3961 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4021 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4081 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4141 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     4201 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     4261 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4321 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4381 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     4441 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4501 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4561 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4621 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4681 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     4741 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4801 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4861 gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4921 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4981 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     5041 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5101 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5161 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5221 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5281 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5341 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5401 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5461 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5521 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5581 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5641 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5701 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5761 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5821 gctggccttt tgctcacat
//
