LOCUS       sequence-325812-        5432 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Direct-expressing EGFP AAV Virus. Alias: AiP995 - 
            pAAV-AiE2010m-minBG-EGFP-WPRE-HGHpA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5432)
  AUTHORS   Graybuck LT, Daigle TL, Sedeno-Cortes AE, Walker M, Kalmbach B, Lenz
            GH, Morin E, Nguyen TN, Garren E, Bendrick JL, Kim TK, Zhou T, 
            Mortrud M, Yao S, Siverts LA, Larsen R, Gore BB, Szelenyi ER, Trader
            C, Balaram P, van Velthoven CTJ, Chiang M, Mich JK, Dee N, Goldy J, 
            Cetin AH, Smith K, Way SW, Esposito L, Yao Z, Gradinaru V, Sunkin 
            SM, Lein E, Levi BP, Ting JT, Zeng H, Tasic B
  TITLE     Enhancer viruses for combinatorial cell-subclass-specific labeling.
  JOURNAL   Neuron. 2021 Mar 23. pii: S0896-6273(21)00159-8. doi: 
            10.1016/j.neuron.2021.03.011.
  PUBMED    33789083
REFERENCE   2  (bases 1 to 5432)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Neuron. 
            2021 Mar 23. pii: S0896-6273(21)00159-8. doi: 
            10.1016/j.neuron.2021.03.011."
COMMENT     Sequence Label: AiP995-pAAV-mscRE10-minBGpromoter-EGFP-WPRE-hGHpA
FEATURES             Location/Qualifiers
     source          1..5432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      809..818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(815..817,818..820,821..1534)
                     /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(815..817,818..820,821..1531)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(860..881)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1121..1140)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1468..1489
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1559..2147
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1612..1632)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2030..2041)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2179..2655
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2378..2397)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2695..2835
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2695..2824
                     /label=AAV2 ITR
     rep_origin      2910..3365
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2997..3016)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3207..3228
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3382..3401)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3501..3523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3561..3579)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3647..3751
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3752..3820,3821..4612)
                     /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(3970..3989)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4783..5371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5272..5291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtatgtgt cttttactct gatcctcctg tttttacctt
      181 ccaagtgctg gaatcacaga catataccac tgtgcatagc atcattacaa tgttatagtt
      241 tttcacacta tgccttgact ttttggaaag gcaaaccacc tcttggattt ctccttcctt
      301 ctctatctct ctctctctct cttcctccct ccgtccctcc atctcttcct ccttcccatt
      361 ttcttctctc cctatttgga cacaatataa aataatttag atgaggtgag ttaaattgtg
      421 aacaaagtat gtgcctatac atggttgtaa atcagcttat caaagtgtaa tattagaaga
      481 atttataaaa atgataaaat tcatactcaa agttctgtgt aaagcaataa tagctttatc
      541 tccttttagt tatcttgagt ctttctatga ctaacaactc cctcataggc atcttaaaga
      601 gcagtaagca taagtagatt ccaaatggga agggagaagt gtgaaccatc actttcatcc
      661 agacttgtag atatatctgc tgcattttca gaaaccagaa acagacagtg ttctttatct
      721 ccattgagtc tagtgtagca acagagctcg ggctgggcat aaaagtcagg gcagagccat
      781 ctattgctta catttgcttc tgggatccgc caccatggtg agcaagggcg aggagctgtt
      841 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
      901 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
      961 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1021 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1081 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1141 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1201 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1261 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1321 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1381 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1441 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1501 gatcactctc ggcatggacg agctgtacaa gtaagaattc gatatcaagc ttatcgataa
     1561 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     1621 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     1681 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     1741 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     1801 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     1861 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     1921 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     1981 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     2041 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     2101 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
     2161 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     2221 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     2281 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     2341 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     2401 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     2461 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     2521 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     2581 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     2641 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
     2701 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     2761 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     2821 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2881 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     2941 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     3001 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3061 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3121 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3181 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3241 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     3301 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3361 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3421 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3481 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3541 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3601 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3661 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3721 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3781 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3841 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3901 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3961 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4021 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4081 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4141 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4201 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4261 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4321 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4381 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4441 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4501 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4561 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4621 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4681 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4741 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4801 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4861 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4921 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4981 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5041 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5101 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5161 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5221 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5281 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5341 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5401 cctggccttt tgctggcctt ttgctcacat gt
//
