LOCUS       sequence-482463-        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  JOURNAL   Unpublished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            "Unpublished"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"
     rep_origin      1..588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     489..508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(600..627)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(651..1466)
                     /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     1374..1393
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1467..1569)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(1570..1601)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     1649..1665
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1649..1665
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1813..2037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2026..2043
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      3352..3361
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3380..3399)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3381..3399)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3444..3647)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        3648..3951
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(4102..4350)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(4462..4710)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(4822..5070)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        complement(5182..5430)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   5471..5611
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5482..5611
                     /label=AAV2 ITR
     polyA_signal    5651..6127
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     5909..5928
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     CDS             complement(join(6186..6896,6897..6899,6900..6902))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6186..6896,6897..6899,6900..6902))
                     /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(6228..6249)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6577..6596
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6836..6857
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          6983..7361
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     primer_bind     complement(7489..7513)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(7510..7713)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        7714..8017
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   8093..8222
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     complement(8410..8427)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8410..8426)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaggt aactggcttc
      121 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      541 gagcatcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      601 gcagaaaggc ccacccgaag gtgagccagg tgattacatt taggtcctca ttagaaaaac
      661 tcatcgagca tcaagtgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
      721 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
      781 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
      841 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
      901 gagaatggca agagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
      961 tcgtcatcaa aatcactcgc accaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     1021 agacgaaata cgcgatcgcc gttaaaagga caattacaaa caggaatcga atgcaaccgg
     1081 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     1141 acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc atcaggagta
     1201 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagcctgacc
     1261 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     1321 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     1381 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cttcaagcaa
     1441 gacgtttccc gttgaatatg gctcatttta gcttccttag ctcctgaaaa tctcgataac
     1501 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     1561 tgccgatcaa gtcaaaagcc tccggtcgga ggcttttgac tttctgctat ggaggtcagg
     1621 tatgatttaa atggtcagta ttgagcctca ggaaacagct atgacatcaa gctgactaga
     1681 taatctagct gatcgtggac cgatcatacg tataatgccg taagatcacg ggtcgcagca
     1741 cagctcgcgg tccagtagtg atcgacactg ctcgatccgc tcgcacccct agggttcttt
     1801 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     1861 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     1921 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     1981 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     2041 aggctgatca gcgggtttaa acgggccctc tagactcgag ttatttccag caaatcagac
     2101 agtaattctt tttaccgcga cgcagtaagg taaaacgacc aaacagacga tcttcttctt
     2161 taaagaagta ttcaggatcg gactgttttt caccgttaat ggtgatggca ttggaggcga
     2221 tagttttacg tgcctgacca cgggaaggtt gcagttcaga atcgaccagt gcctgcatca
     2281 ggtctgcgcc cttttccatc tcaaccatcg gtacgccgtc ctgcgccagc tgttcgaagt
     2341 ccgcttcact cagcgcactc aaagaaccgc tgaacaggca ttcggtaata cgttttgccg
     2401 cctgtaaacc ttcttcaccg tgaaccagac gagtcacctg ctccgccagt acatactggg
     2461 cgcgcggtgc tttaccgctg tttttatctt cttcttccag ggcgttgatc tcttcaatgc
     2521 tcataaaggt gaagaacttc aggaagcggt aaacgtcggc acgcgcagtg ttgatccaga
     2581 actggtagaa tttgtacggg ctggttttct tcggatctaa ccagactgcg ccgccttcag
     2641 ttttaccaaa tttggtgcca tctgctttag tgatcagcgg aacggtcagg ccaaacacct
     2701 gattctgatg cagacgacgg gtcaggtcga taccagaagt gatgttaccc cactggtcag
     2761 aaccaccaat ttgcagcacc acaccgtact gtttgttaca acaggcataa caataaccct
     2821 gcaacaggtt gtaggaaaac tcagtgaacg aaatcccctg atcttcacgg ttgagacgct
     2881 gcttaaccgc ttctttgttg atcatctggt taacggagaa gtgtttgcca atatcgcgca
     2941 ggaaggtcag cacattcata ttgccgaacc agtcatagtt gttcgccgcg atagcagagt
     3001 tttctccaca gtcgaaatcg aggaacgggg caacctgctt acggattttg tccacccact
     3061 cctgaacagt ttcttcggtg ttcagcttac gctcggcagc tttgaagctc gggtcgccaa
     3121 tcagacccgt cgcgccgcct acaaccgcaa ccggcttgtg gcccgcctgc tggaagcgtt
     3181 tcaggcataa caatggaaca agatgcccca aatgcaagct gtcagcggta ggatcgaagc
     3241 cgcacccgag cgcgatcggg ccttgcgcca gtcgctctgc taacgcttcc tcgtccgtca
     3301 cctgggctac cagcccccgc tcttgcaatt gtttaatcaa gttactgctt gccatggtgg
     3361 cgctagccag cttgggtctc cctatagtga gtcgtattaa tttcgataag ccagtaagca
     3421 gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg tacacgccta
     3481 ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt
     3541 tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag
     3601 tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg
     3661 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
     3721 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
     3781 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
     3841 tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg
     3901 gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc
     3961 atatatgggc tatgaactaa tgaccccgta attgattact attaataact agcaaaaaat
     4021 gggacgggaa ggattcgaac cttcgaagtc tgtgacggca gatttagagt ctgctccctt
     4081 tggccgctcg ggaaccgtcc cggtgtttcg tcctttccac aagatatata aagccaagaa
     4141 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     4201 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     4261 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     4321 atatgaagga atcatgggaa ataggccctc ttcctgcccg aggtacccct agcaaaaaat
     4381 gggacgggaa ggattcgaac cttcgaagtc tgtgacggca gatttagagt ctgctccctt
     4441 tggccgctcg ggaaccgtcc cggtgtttcg tcctttccac aagatatata aagccaagaa
     4501 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     4561 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     4621 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     4681 atatgaagga atcatgggaa ataggccctc ttcctgcccg aggtacccct agcaaaaaat
     4741 gggacgggaa ggattcgaac cttcgaagtc tgtgacggca gatttagagt ctgctccctt
     4801 tggccgctcg ggaaccgtcc cggtgtttcg tcctttccac aagatatata aagccaagaa
     4861 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     4921 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     4981 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     5041 atatgaagga atcatgggaa ataggccctc ttcctgcccg aggtacccct agcaaaaaat
     5101 gggacgggaa ggattcgaac cttcgaagtc tgtgacggca gatttagagt ctgctccctt
     5161 tggccgctcg ggaaccgtcc cggtgtttcg tcctttccac aagatatata aagccaagaa
     5221 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     5281 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     5341 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     5401 atatgaagga atcatgggaa ataggccctc ttcctgcccg aggtacccct aggggtgcga
     5461 gcggatcgag cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
     5521 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
     5581 ggagtggcca actccatcac taggggttcc tgcggccgct cggtccgcac gtggttacct
     5641 acaaaatcag aaggacaggg aagggagcag tggttcacgc ctgtaatccc agcaatttgg
     5701 gaggccaagg tgggtagatc acctgagatt aggagttgga gaccagcctg gccaatatgg
     5761 tgaaaccccg tctctaccaa aaaaacaaaa attagctgag cctggtcatg catgcctgga
     5821 atcccaacaa ctcgggaggc tgaggcagga gaatcgcttg aacccaggag gcggagattg
     5881 cagtgagcca agattgtgcc actgcactcc agcttggttc ccaatagacc ccgcaggccc
     5941 tacaggttgt cttcccaact tgccccttgc tccataccac ccccctccac cccataatat
     6001 tatagaagga cacctagtca gacaaaatga tgcaacttaa ttttattagg acaaggctgg
     6061 tgggcactgg agtggcaact tccagggcca ggagaggcac tggggagggg tcacagggat
     6121 gccacccgta gatctctcga gcagcgctgc tcgaggcaag cttacttgag ctcgagatct
     6181 gagtacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     6241 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     6301 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     6361 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     6421 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     6481 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     6541 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     6601 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     6661 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     6721 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     6781 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     6841 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     6901 attcagaatt caatcgatgt tcgaatccca attctttgcc aaagtgatgg gccagcacac
     6961 agaccagcac gttgcccagg agctgtggga ggaagataag aggtatgaac atgattagca
     7021 aaagggccta gcttggactc agaataatcc agccttatcc caaccataaa ataaaagcag
     7081 aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttacatc agttacaatt
     7141 tatatgcaga aatatttata tgcagaaata ttgctattgc cttaacccag aaattatcac
     7201 tgttattctt tagaatggtg caaagaggca tgatacattg tatcattatt gccctgaaag
     7261 aaagagatta gggaaagtat tagaaataag ataaacaaaa aagtatatta aaagaagaaa
     7321 gcattttttg tgggcctata gactctatag gcggtactta cgtcactctt ggcacgggga
     7381 atccgcgttc caatgcaccg ttcccggccg ggattcgaat ccgcggaggc tggatcggtc
     7441 ccggtgtctt ctatggaggt caaaacagcg tggatggcgt ctccaggcga tctgacggtt
     7501 cactaaacga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     7561 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     7621 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     7681 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     7741 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     7801 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     7861 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     7921 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     7981 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     8041 aactaatgac cccgtaattg attactatta ataactagac gcgtgcggcc gcaggaaccc
     8101 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     8161 ccaaaggtcg cccgacgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     8221 ggtatttgcg gccgccctag cggatcgacg agagcagcgc gactggatct gtcgcccgtc
     8281 tcaaacgcaa ccctccggcg gtcgcatatc attcaggacg agcctcagac tccagcgtaa
     8341 ctggactgca atcaactcac tggctcacct tccggtccac gatcagctag aatcaagctg
     8401 actagataaa ctggccgtcg ttttacacgg gtgggccttt cttcggtaga aaatcaaagg
     8461 atcttc
//
