LOCUS       sequence-225358-        5925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV genome containing miRNA target sequence (TS) 708-5p to reduce
            expression of the fluorescent protein eYFP in neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5925)
  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 5925)
  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-miR708-5p-TS
FEATURES             Location/Qualifiers
     source          1..5925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     439..461
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     523..542
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(585..587,588..590,591..1304)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(630..651)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(891..910)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1238..1259
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1418..2006
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1471..1491)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1889..1900)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2038..2514
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2237..2256)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2554..2694
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2554..2683
                     /label=AAV2 ITR
     rep_origin      2769..3224
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2856..2875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3066..3087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3241..3260)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3360..3382
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3420..3438)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3506..3610
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3611..3679,3680..4471)
                     /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(3829..3848)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4642..5230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5131..5150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5292..5421
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        5501..5804
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
       61 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      121 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
      181 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
      241 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
      301 gctccgggag cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
      361 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
      421 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
      481 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
      541 tgctgtctca tcattttggc aaagaattgc ggcccaacgg taccatggtg agcaagggcg
      601 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
      661 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
      721 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg
      781 gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
      841 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
      901 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
      961 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1021 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1081 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1141 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     1201 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1261 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagaattc accggtccca
     1321 gctagattgt aagctcctta ctgtacccag ctagattgta agctccttga ctcacccagc
     1381 tagattgtaa gctccttcgc cggcgaagct tatcgataat caacctctgg attacaaaat
     1441 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     1501 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     1561 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     1621 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     1681 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     1741 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     1801 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     1861 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     1921 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     1981 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg
     2041 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     2101 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     2161 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     2221 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     2281 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     2341 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     2401 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     2461 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     2521 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     2581 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     2641 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     2701 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     2761 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2821 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     2881 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2941 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     3001 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3061 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     3121 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     3181 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     3241 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     3301 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3361 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     3421 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3481 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3541 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3601 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3661 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3721 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3781 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3841 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3901 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3961 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4021 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4081 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4141 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4201 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4261 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4321 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4381 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4441 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5281 tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc
     5341 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5401 actccatcac taggggttcc tgcggccgca cgcgtaacac tagttattaa tagtaatcaa
     5461 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     5521 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     5581 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     5641 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     5701 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     5761 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     5821 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     5881 ttttttaatt attttgtgca gcgatggggg cggggggggg gggcg
//
