LOCUS       sequence-487066-        8466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AAV2 transgene (EGFP) and EcYRS/tRNA for ncAA incorporation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8466)
  TITLE     An expanded toolbox for precise chemical editing of the 
            adeno-associated virus capsid
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8466)
  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            "Unpublished"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: pIDTsmart-EcYRS-4xYtR-ITR-GFP
FEATURES             Location/Qualifiers
     source          1..8466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6..22
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   210..339
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        415..718
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        719..922
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     919..943
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1071..1449
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             join(1530..1532,1533..1535,1536..2246)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1530..1532,1533..1535,1536..2246)
                     /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(1575..1596)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1836..1855)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2183..2204
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2305..2781
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2504..2523)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2821..2961
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2821..2950
                     /label=AAV2 ITR
     promoter        3002..3250
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3362..3610
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        3722..3970
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        4082..4330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     enhancer        4481..4784
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4785..4988
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5033..5052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5033..5051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5071..5080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6389..6406)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6395..6619
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6767..6783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6767..6783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      6831..6862
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        6863..6965
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             6966..7781
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(7039..7058)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      7805..7832
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(7844..8431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7924..7943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtgtaaa acgacggcca gtttatctag tcagcttgat tctagctgat cgtggaccgg
       61 aaggtgagcc agtgagttga ttgcagtcca gttacgctgg agtctgaggc tcgtcctgaa
      121 tgatatgcga ccgccggagg gttgcgtttg agacgggcga cagatccagt cgcgctgctc
      181 tcgtcgatcc gctagggcgg ccgcaaatac ctgcaggcag ctgcgcgctc gctcgctcac
      241 tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc
      301 gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg cgtctagtta
      361 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      421 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      481 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      541 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      601 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      661 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      721 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      781 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      841 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      901 ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
      961 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgcg gattcgaatc
     1021 ccggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac gtaagtaccg
     1081 cctatagagt ctataggccc acaaaaaatg ctttcttctt ttaatatact tttttgttta
     1141 tcttatttct aatactttcc ctaatctctt tctttcaggg caataatgat acaatgtatc
     1201 atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa ggcaatagca
     1261 atatttctgc atataaatat ttctgcatat aaattgtaac tgatgtaaga ggtttcatat
     1321 tgctaatagc agctacaatc cagctaccat tctgctttta ttttatggtt gggataaggc
     1381 tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc tcttatcttc
     1441 ctcccacagc tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt tggcaaagaa
     1501 ttgggattcg aacatcgatt gaattctgaa tggtgagcaa gggcgaggag ctgttcaccg
     1561 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1621 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1681 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1741 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1801 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1861 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1921 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1981 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2041 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2101 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2161 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2221 ctctcggcat ggacgagctg tacaagtact cagatctcga gctcaagtaa gcttgcctcg
     2281 agcagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     2341 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     2401 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     2461 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     2521 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     2581 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     2641 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     2701 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     2761 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     2821 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2881 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2941 gagcgcgcag ctgcctgcag gctcgatccg ctcgcacccc taggggtacc tcgggcagga
     3001 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     3061 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     3121 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     3181 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     3241 acgaaacacc gggacggttc ccgagcggcc aaagggagca gactctaaat ctgccgtcac
     3301 agacttcgaa ggttcgaatc cttcccgtcc cattttttgc taggggtacc tcgggcagga
     3361 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     3421 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     3481 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     3541 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     3601 acgaaacacc gggacggttc ccgagcggcc aaagggagca gactctaaat ctgccgtcac
     3661 agacttcgaa ggttcgaatc cttcccgtcc cattttttgc taggggtacc tcgggcagga
     3721 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     3781 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     3841 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     3901 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     3961 acgaaacacc gggacggttc ccgagcggcc aaagggagca gactctaaat ctgccgtcac
     4021 agacttcgaa ggttcgaatc cttcccgtcc cattttttgc taggggtacc tcgggcagga
     4081 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     4141 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     4201 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     4261 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     4321 acgaaacacc gggacggttc ccgagcggcc aaagggagca gactctaaat ctgccgtcac
     4381 agacttcgaa ggttcgaatc cttcccgtcc cattttttgc tagttattaa tagtaatcaa
     4441 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     4501 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     4561 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     4621 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     4681 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     4741 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     4801 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     4861 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     4921 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     4981 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     5041 ctcactatag ggagacccaa gctggctagc gccaccatgg caagcagtaa cttgattaaa
     5101 caattgcaag agcgggggct ggtagcccag gtgacggacg aggaagcgtt agcagagcga
     5161 ctggcgcaag gcccgatcgc gctcgggtgc ggcttcgatc ctaccgctga cagcttgcat
     5221 ttggggcatc ttgttccatt gttatgcctg aaacgcttcc agcaggcggg ccacaagccg
     5281 gttgcggttg taggcggcgc gacgggtctg attggcgacc cgagcttcaa agctgccgag
     5341 cgtaagctga acaccgaaga aactgttcag gagtgggtgg acaaaatccg taagcaggtt
     5401 gccccgttcc tcgatttcga ctgtggagaa aactctgcta tcgcggcgaa caactatgac
     5461 tggttcggca atatgaatgt gctgaccttc ctgcgcgata ttggcaaaca cttctccgtt
     5521 aaccagatga tcaacaaaga agcggttaag cagcgtctca accgtgaaga tcaggggatt
     5581 tcgttcactg agttttccta caacctgttg cagggttatt gttatgcctg ttgtaacaaa
     5641 cagtacggtg tggtgctgca aattggtggt tctgaccagt ggggtaacat cacttctggt
     5701 atcgacctga cccgtcgtct gcatcagaat caggtgtttg gcctgaccgt tccgctgatc
     5761 actaaagcag atggcaccaa atttggtaaa actgaaggcg gcgcagtctg gttagatccg
     5821 aagaaaacca gcccgtacaa attctaccag ttctggatca acactgcgcg tgccgacgtt
     5881 taccgcttcc tgaagttctt cacctttatg agcattgaag agatcaacgc cctggaagaa
     5941 gaagataaaa acagcggtaa agcaccgcgc gcccagtatg tactggcgga gcaggtgact
     6001 cgtctggttc acggtgaaga aggtttacag gcggcaaaac gtattaccga atgcctgttc
     6061 agcggttctt tgagtgcgct gagtgaagcg gacttcgaac agctggcgca ggacggcgta
     6121 ccgatggttg agatggaaaa gggcgcagac ctgatgcagg cactggtcga ttctgaactg
     6181 caaccttccc gtggtcaggc acgtaaaact atcgcctcca atgccatcac cattaacggt
     6241 gaaaaacagt ccgatcctga atacttcttt aaagaagaag atcgtctgtt tggtcgtttt
     6301 accttactgc gtcgcggtaa aaagaattac tgtctgattt gctggaaata actcgagtct
     6361 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     6421 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6481 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6541 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     6601 gatgcggtgg gctctatggc ttctgaggcg gaaagaaccc taggggtgcg agcggatcga
     6661 gcagtgtcga tcactactgg accgcgagct gtgctgcgac ccgtgatctt acggcattat
     6721 acgtatgatc ggtccacgat cagctagatt atctagtcag cttgatgtca tagctgtttc
     6781 ctgaggctca atactgacca tttaaatcat acctgacctc catagcagaa agtcaaaagc
     6841 ctccgaccgg aggcttttga cttgatcggc acgtaagagg ttccaacttt caccataatg
     6901 aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag
     6961 ctaaaatgag ccatattcaa cgggaaacgt cttgcttgaa gccgcgatta aattccaaca
     7021 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     7081 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     7141 gtagcgttgc caatgatgtt acagatgaga tggtcaggct aaactggctg acggaattta
     7201 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     7261 ctgcgatccc agggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     7321 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     7381 gtccttttaa cggcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     7441 gtttggttgg tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     7501 ggaaagaaat gcataaactc ttgccattct caccggattc agtcgtcact catggtgatt
     7561 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     7621 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     7681 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     7741 ataaattgca gtttcacttg atgctcgatg agtttttcta atgaggacct aaatgtaatc
     7801 acctggctca ccttcgggtg ggcctttctg cgttgctggc gtttttccat aggctccgcc
     7861 cccctgacga gcatcacaaa aatcgatgct caagtcagag gtggcgaaac ccgacaggac
     7921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     8041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     8101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     8161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     8221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     8281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt tacctcggaa aaagagttgg
     8341 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     8401 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgattttct accgaagaaa
     8461 ggccca
//
