LOCUS       sequence-225354-        5816 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  An AAV genome containing miRNA target sequence (TS) 204-5p to reduce
            expression of the fluorescent protein eYFP in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5816)
  AUTHORS   Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ,
            Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, 
            Gradinaru V
  TITLE     Systemic AAV vectors for widespread and targeted gene delivery in 
            rodents.
  JOURNAL   Nat Protoc. 2019 Feb;14(2):379-414. doi: 10.1038/s41596-018-0097-3.
  PUBMED    30626963
REFERENCE   2  (bases 1 to 5816)
  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"; doi: "10"; journalName: 
            "Nat Protoc"; date: "2019-02"; volume: "14"; issue: "2"; pages: 
            "379-414"
COMMENT     Sequence Label: pAAV-CAG-eYFP-3x-miR204-5p-TS
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     445..467
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     529..548
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(591..593,594..596,597..1310)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(636..657)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(897..916)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1244..1265
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1421..2009
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1474..1494)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1892..1903)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2041..2517
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2240..2259)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2557..2697
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2557..2686
                     /label=AAV2 ITR
     rep_origin      2772..3227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2859..2878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3069..3090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3244..3263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3363..3385
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3423..3441)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3509..3613
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3614..3682,3683..4474)
                     /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(3832..3851)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4645..5233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5134..5153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5295..5424
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5504..5807
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
       61 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      121 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
      181 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
      241 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
      301 tgaggggctc cgggagcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc
      361 cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc
      421 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
      481 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
      541 ttattgtgct gtctcatcat tttggcaaag aattgcggcc caacggtacc atggtgagca
      601 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      661 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      721 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
      781 ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact
      841 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
      901 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
      961 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1021 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1081 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1141 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     1201 accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1261 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gaattcaccg
     1321 gtaggcatag gatgacaaag ggaatatcac aggcatagga tgacaaaggg aactcagaag
     1381 gcataggatg acaaagggaa cgccggcgaa gcttatcgat aatcaacctc tggattacaa
     1441 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     1501 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     1561 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     1621 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     1681 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     1741 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     1801 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat
     1861 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     1921 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     1981 tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac
     2041 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
     2101 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
     2161 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca
     2221 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg
     2281 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
     2341 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg
     2401 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct
     2461 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg
     2521 attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg
     2581 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     2641 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     2701 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     2761 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2821 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     2881 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2941 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     3001 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3061 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     3121 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     3181 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     3241 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     3301 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3361 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     3421 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     3481 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3541 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3601 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3661 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3721 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3781 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3841 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3901 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3961 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     4021 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     4081 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     4141 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     4201 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4261 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4321 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4381 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4441 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4501 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4561 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     5101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     5161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     5221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     5281 ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc
     5341 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     5401 ccaactccat cactaggggt tcctgcggcc gcacgcgtaa cactagttat taatagtaat
     5461 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     5521 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     5581 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     5641 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     5701 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     5761 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtg
//
