LOCUS       sequence-481562-        8342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AAV2 transgene (EGFP) and EcWRS/tRNA for ncAA incorporation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8342)
  TITLE     An expanded toolbox for precise chemical editing of the 
            adeno-associated virus capsid
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8342)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-EcWRS-4xWtR-ITR-GFP
FEATURES             Location/Qualifiers
     source          1..8342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     710..734
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          862..1240
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             join(1321..1323,1324..1326,1327..2037)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1321..1323,1324..1326,1327..2037)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1366..1387)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1627..1646)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1974..1995
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2096..2572
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2295..2314)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2612..2752
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2612..2741
                     /label=AAV2 ITR
     promoter        2787..3035
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3135..3383
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3483..3731
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3831..4079
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     enhancer        4224..4527
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4606..4657
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     primer_bind     4702..4721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4702..4720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5834..5851)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5840..6064
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6069..6524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6156..6175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6366..6387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6550..6654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6655..6723,6724..7515)
                     /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(6873..6892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7686..8274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8175..8194
                     /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 gcgtctagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
      721 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      781 gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg
      841 attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat
      901 gctttcttct tttaatatac ttttttgttt atcttatttc taatactttc cctaatctct
      961 ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa
     1021 cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata
     1081 taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca
     1141 ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct
     1201 tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt
     1261 ctgtgtgctg gcccatcact ttggcaaaga attgggattc gaacatcgat tgaattctga
     1321 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1381 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1441 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1501 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1561 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1621 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1681 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1741 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1801 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1861 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1921 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1981 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtac
     2041 tcagatctcg agctcaagta agcttgcctc gagcagcgct gctcgagaga tctacgggtg
     2101 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     2161 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     2221 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     2281 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     2341 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     2401 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     2461 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     2521 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     2581 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     2641 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     2701 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggctcgatcc
     2761 gctcgcaccc ctaggtcggg caggaagagg gcctatttcc catgattcct tcatatttgc
     2821 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2881 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2941 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3001 tggctttata tatcttgtgg aaaggacgaa acaccagggg cgtagttcaa ttggtagagc
     3061 accggtcttc aaaaccgggt gttgggagtt cgagtctctc cgcccctgcc attttttgct
     3121 aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca
     3181 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3241 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3301 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3361 tcttgtggaa aggacgaaac accaggggcg tagttcaatt ggtagagcac cggtcttcaa
     3421 aaccgggtgt tgggagttcg agtctctccg cccctgccat tttttgctag gtcgggcagg
     3481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     3541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     3601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     3661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     3721 gacgaaacac caggggcgta gttcaattgg tagagcaccg gtcttcaaaa ccgggtgttg
     3781 ggagttcgag tctctccgcc cctgccattt tttgctaggt cgggcaggaa gagggcctat
     3841 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
     3901 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     3961 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     4021 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacacca
     4081 ggggcgtagt tcaattggta gagcaccggt cttcaaaacc gggtgttggg agttcgagtc
     4141 tctccgcccc tgccattttt tgctagttat taatagtaat caattacggg gtcattagtt
     4201 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     4261 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     4321 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     4381 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     4441 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     4501 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     4561 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgc aaatgggcgg
     4621 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     4681 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg
     4741 tttaaactta agcttgccgc caccatgact aagcccatcg tttttgctgg cgcacagccc
     4801 tcaggtgaat tgaccattgg taactacatg ggtgcgctgc gtcagtgggt aaacatgcag
     4861 gatgactacc attgcattta ctgtatcgtt gaccaacacg cgatcaccgt gcgccaggat
     4921 gcacagaagc tgcgtaaagc gacgctggat acgctggcct tgtatctggc ttgtggtatc
     4981 gatcctgaga aaagcaccat ttttgttcag tcccacgtgc cggaacatgc acagttaggc
     5041 tgggcactga actgctatac ctacttcggc gaactgagtc gcatgacgca gtttaaagat
     5101 aaatctgcgc gttatgccga gaacatcaac gctggtctgt ttgactatcc ggtgctgatg
     5161 gcagcggaca tcctgctgta tcaaactaat ctgggtcctt gtggtgaaga ccagaaacag
     5221 cacctcgaac tgagccgcga tattgcccag cgtttcaacg cgctgtatgg cgagatcttt
     5281 aaggtgccgg agccgtttat tccgaaatct ggcgcgcgcg taatgtcgct gctggagccg
     5341 accaagaaga tgtccaagtc tgacgataat cgcaataacg ttatcggcct gctggaagat
     5401 ccgaaatcgg tagtgaagaa aatcaaacgt gcggtcactg actccgacga gccgccggta
     5461 gttcgctacg atgtgcagaa caaagcgggc gtttccaacc tgttggatat cctttcagcg
     5521 gtaacgggcc agagcatccc agaactggaa aaacagttcg aaggcaagat gtatggtcat
     5581 ctgaaaggtg aagtggctga tgccgtttcc ggtatgctga ctgaattgca ggaacgctat
     5641 caccgtttcc gcaacgatga agccttcctg caacaggtga tgaaagatgg cgcggaaaaa
     5701 gccagcgcgc acgcttcccg tacgctaaaa gcggtgtacg aagcgattgg ttttgtggcg
     5761 aagccgtaag aattcaacgc gttaagtcga ctttaactcg agtctagagg gcccgtttaa
     5821 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     5881 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     5941 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     6001 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     6061 atggtaccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     6121 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     6181 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     6241 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     6301 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6361 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     6421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     6481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     6541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6841 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     7021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     7081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     7141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     7201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     7261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     7321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     7381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7561 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7621 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7681 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7741 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7801 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7861 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7921 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7981 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     8041 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     8101 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     8161 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     8221 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     8281 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgcggc
     8341 cg
//
