LOCUS       sequence-413643-        5791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5791)
  AUTHORS   Huang Q, Chen AT, Chan KY, Sorensen H, Barry AJ, Azari B, Zheng Q, 
            Beddow T, Zhao B, Tobey IG, Moncada-Reid C, Eid FE, Walkey CJ, 
            Ljungberg MC, Lagor WR, Heaney JD, Chan YA, Deverman BE
  TITLE     Targeting AAV vectors to the central nervous system by engineering 
            capsid-receptor interactions that enable crossing of the blood-brain
            barrier.
  JOURNAL   PLoS Biol. 2023 Jul 19;21(7):e3002112. doi: 
            10.1371/journal.pbio.3002112. eCollection 2023 Jul.
  PUBMED    37467291
REFERENCE   2  (bases 1 to 5791)
  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: "PLoS 
            Biol."; date: "2023-07-19"; volume: "21(7):e3002112. doi"; pages: " 
            10.1371/journal.pbio.3002112. eCollection 2023 Jul"
COMMENT     Sequence Label: AAV-GfABC1D-NLS-GFP-WPRE-synpA-L1-R2
FEATURES             Location/Qualifiers
     source          1..5791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             889..909
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(919..921,922..924,925..1638)
                     /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(919..921,922..924,925..1635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(964..985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1225..1244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1572..1593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1655..2243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1708..1728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2126..2137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2260..2308
                     /note="synthetic polyadenylation signal"
     promoter        2312..2560
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     promoter        2682..2930
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   3054..3194
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3054..3183
                     /label=AAV2 ITR
     rep_origin      3269..3724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3356..3375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3566..3587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3741..3760)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3860..3882
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3920..3938)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4006..4110
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4111..4179,4180..4971)
                     /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"
     rep_origin      5142..5730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5631..5650
                     /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 gcggcctcta gaacgcgtaa catatcctgg tgtggagtag gggacgctgc
      181 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      241 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      301 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      361 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      421 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      481 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      541 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      601 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      661 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
      721 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgtatc ccaggagcga
      781 gcagagccag agcaggttgg agaggagacg catcacctct gctgcttgac cggtgccacc
      841 atgtctagtg atgatgaggc tactgctgac tctcaacatt ctactcctcc aaaaaagaag
      901 agaaaggtag aagaccccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      961 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1021 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1081 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     1141 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1201 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1261 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1321 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1381 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1441 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1501 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1561 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1621 gacgagctgt acaagtaaga attcgcttat cgataatcaa cctctggatt acaaaatttg
     1681 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     1741 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     1801 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     1861 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     1921 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     1981 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     2041 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg
     2101 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     2161 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     2221 ctccctttgg gccgcctccc cgcatcgata ccgaagctta ataaaatatc tttattttca
     2281 ttacatctgt gtgttggttt tttgtgtgct cgagggccta tttcccatga ttccttcata
     2341 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     2401 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2461 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2521 tttcttggct ttatatatct tgtggaaagg acgaaacacc gcctctagag tcgcagatcc
     2581 tcgtttaagt actctgtgct ggaaacagca cagaatctac ttaaacaagg caaaatgccg
     2641 tgtttatctc gtcaacttgt tggcgagatt tttttggtac cgagggccta tttcccatga
     2701 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2761 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2821 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2881 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gcgacctgca
     2941 gcccaagcta gagtttaagt actctgtgct ggaaacagca cagaatctac ttaaacaagg
     3001 caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttttgcggc cgcaggaacc
     3061 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     3121 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     3181 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3241 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     3301 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     3361 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3421 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3481 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3541 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3601 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3661 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3721 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3781 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3841 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3901 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3961 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4021 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4081 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4141 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4201 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4261 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4321 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4381 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4441 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4501 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4561 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4621 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4681 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4741 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4801 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     4861 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4921 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4981 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5041 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5101 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5161 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5221 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5281 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5341 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5401 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5461 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5521 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5581 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5641 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5701 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5761 ctggcctttt gctggccttt tgctcacatg t
//
