LOCUS       sequence-458356-        5172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for producing AAV-CMV-eGFP viruses with the AAVtri 
            triple-plasmid system. This system combines the use of the 
            mini-pHelper-1.0 and AAV helper plasmids carrying different AAV 
            serotypes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5172)
  AUTHORS   Yang R, Tran NT, Chen T, Cui M, Wang Y, Sharma T, Liu Y, Zhang J, 
            Yuan X, Zhang D, Chen C, Shi Z, Wang L, Dai Y, Zaidi H, Liang J, 
            Chen M, Jaijyan D, Hu H, Wang B, Xu C, Hu W, Gao G, Yu D, Tai PWL, 
            Wang Q
  TITLE     AAVone: A cost-effective, single-plasmid solution for efficient AAV 
            production with reduced DNA impurities.
  JOURNAL   Mol Ther Nucleic Acids. 2025 May 14;36(2):102563. doi: 
            10.1016/j.omtn.2025.102563. eCollection 2025 Jun 10.
  PUBMED    40503174
REFERENCE   2  (bases 1 to 5172)
  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: "Mol Ther 
            Nucleic Acids."; date: "2025-05-14"; volume: "36(2):102563. doi"; 
            pages: " 10.1016/j.omtn.2025.102563. eCollection 2025 Jun 10"
COMMENT     Sequence Label: pAAVtri-CMV-eGFP
FEATURES             Location/Qualifiers
     source          1..5172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        134..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          717..813
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     regulatory      818..827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(824..826,827..829,830..1543)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(824..826,827..829,830..1540)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(869..890)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1130..1149)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1477..1498
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1568..2156
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1621..1641)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2039..2050)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2188..2664
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2387..2406)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2673..2813
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2673..2802
                     /label=AAV2 ITR
     rep_origin      2888..3343
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2975..2994)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3185..3206
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3369..3473
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3474..4268
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3474..4268
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3528..3547)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4138..4157
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      4439..5027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4928..4947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg
      121 cggccgcacg cgtacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc acctgcgtaa
      721 gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa
      781 ctgctcctca gtggatgttg cctttacttc taggcgcgcc accatggtga gcaagggcga
      841 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
      901 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
      961 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     1021 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     1081 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     1141 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     1201 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     1261 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     1321 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     1381 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     1441 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     1501 cgccgccggg atcactctcg gcatggacga gctgtacaag taacgtacgg atatcaagct
     1561 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     1621 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     1681 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     1741 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     1801 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     1861 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     1921 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     1981 gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     2041 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     2101 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     2161 ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct
     2221 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     2281 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     2341 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     2401 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     2461 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     2521 taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac
     2581 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     2641 accactgctc ccttccctgt ccttttaatt aaaggaaccc ctagtgatgg agttggccac
     2701 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     2761 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     2821 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     2881 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2941 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     3001 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3061 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     3121 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3181 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3241 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3301 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3361 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3421 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgattg
     3481 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3541 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3601 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     3661 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtactcgacg
     3721 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3781 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3841 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgaga
     3901 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3961 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4021 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4081 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4141 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4201 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4261 tcttctgact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4321 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4381 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4441 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4501 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4561 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4621 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4681 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4741 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4801 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4861 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4921 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4981 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5041 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5101 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat catatggttt
     5161 aaacgttggc ag
//
