LOCUS       sequence-385161-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Astrocytic expression of membrane-targeted V5 spaghetti monster 
            reporter in AAV; astrocyte specificity enhanced with 4x6T miRNA 
            targeting cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  AUTHORS   Gleichman AJ, Kawaguchi R, Sofroniew MV, Carmichael ST
  TITLE     A toolbox of astrocyte-specific, serotype-independent 
            adeno-associated viral vectors using microRNA targeting sequences.
  JOURNAL   Nat Commun. 2023 Nov 16;14(1):7426. doi: 10.1038/s41467-023-42746-w.
  PUBMED    37973910
REFERENCE   2  (bases 1 to 6866)
  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: "Nat 
            Commun."; date: "2023-11-16"; volume: "14(1):7426. doi"; pages: " 
            10.1038/s41467-023-42746-w"
COMMENT     Sequence Label: AAV-GfaABC1D-lck-smV5-4x6T-WPRE
FEATURES             Location/Qualifiers
     source          1..6866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   673..802
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1522..1531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1663..1672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1672..1713
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1723..1764
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1774..1815
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2347..2388
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2398..2439
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2458..2499
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2509..2550
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2764..2805
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2815..2856
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2866..2907
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    3630..4218
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3683..3703)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4101..4112)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4276..4752
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4475..4494)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4801..4941
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4801..4930
                     /label=AAV2 ITR
     rep_origin      5016..5471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5103..5122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5313..5334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5488..5507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5607..5629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5667..5685)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5753..5857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5858..5926,5927..6718)
                     /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"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      721 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      781 aactccatca ctaggggttc ctgcggccgc acgcgtacat atcctggtgt ggagtagggg
      841 acgctgctct gacagaggct cgggggcctg agctggctct gtgagctggg gaggaggcag
      901 acagccaggc cttgtctgca agcagacctg gcagcattgg gctggccgcc ccccagggcc
      961 tcctcttcat gcccagtgaa tgactcacct tggcacagac acaatgttcg gggtgggcac
     1021 agtgcctgct tcccgccgca ccccagcccc cctcaaatgc cttccgagaa gcccattgag
     1081 cagggggctt gcattgcacc ccagcctgac agcctggcat cttgggataa aagcagcaca
     1141 gccccctagg ggctgccctt gctgtgtggc gccaccggcg gtggagaaca aggctctatt
     1201 cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc atggacagtg ggtggcaggg
     1261 ggggagagga gggctgtctg cttcccagaa gtccaaggac acaaatgggt gaggggagag
     1321 ctctccccat agctgggctg cggcccaacc ccaccccctc aggctatgcc agggggtgtt
     1381 gccaggggca cccgggcatc gccagtctag cccactcctt cataaagccc tcgcatccca
     1441 ggagcgagca gagccagagc aggttggaga ggagacgcat cacctccgct gctcgcttaa
     1501 ttaactaccg gactcagatc tgccaccatg ggctgtggct gcagctcaaa ccctgaagat
     1561 gactggatgg agaacattga cgtgtgtgag aactgccatt atcccccgcc gaagcttcga
     1621 attctgcagt cgacggtacc gcgggcccgg gatccaccgg tcgccaccat gggaaaacct
     1681 ataccgaacc ccctccttgg acttgatagt actcagcagc agggtaagcc cattccaaac
     1741 ccacttctcg gtctggatag cacacagcaa caaggtaagc ctatccctaa ccctctgttg
     1801 ggcctggact ccacgggcca aggtgtgagt aagggcgaag aactgtttac cggcgtggtc
     1861 ccaattctgg ttgagctcga cggtgacgtt aacggacaca agtttagtgt gcgcggagaa
     1921 ggagagggag atgccaccaa cggaaaattg acgctgaagt ttatatgcac cacgggaaag
     1981 ctgccggtgc cgtggccgac cttggttaca accctgggtg gcggagtcca gtgtttcagc
     2041 cgttacccag accacatgaa gcagcatgac ttctttaaga gtgctatgcc ggagggatac
     2101 gtgcaggaac ggacgatcag tttcaaagat gacggtactt acaagacgcg agccgaggtg
     2161 aagttcgagg gagacacctt ggtgaacagg atagaactta agggaattga tttcaaggaa
     2221 gacggtaata ttctgggtca caagctggaa tacaacttta acagccataa cgtctacatt
     2281 actgccgaca agcagaagaa cggcataaag gcgaatttca aaattcggca caacgtggaa
     2341 ggtggtggca agccgatccc gaatcccctg ctgggcttgg atagcacgca gcaacaggga
     2401 aagcccatcc ctaacccctt gctgggactc gacagcaccg gaggccagca aggcggtggc
     2461 aaaccgatac ccaatcccct gctcggcctg gatagcaccc agcagcaagg caagccaatc
     2521 cccaaccctc tgctgggcct cgactcgaca ggtggcgatg gaagtgttca actggcggac
     2581 cactatcagc agaatacgcc gatcggagat ggccccgtgc tgttgccgga taatcactat
     2641 ctgtcgactc agagcgttct gagtaaggac ccaaacgaaa aacgagatca catggtgttg
     2701 ttggagttcg tgaccgccgc cggcattacg ttgggaatgg acgagctgta caagggccaa
     2761 ggaggcaagc ccataccaaa tcccttgctg ggcctcgaca gcactcagca acaaggaaag
     2821 ccaataccaa atccactgtt gggcctcgac agtacgcaac agcagggcaa gcccatcccg
     2881 aacccgttgc tgggactgga tagtacctaa aataggtaaa gcggccgcga ctctagatca
     2941 tatgggcatt caccgcgtgc cttactacgc gtattcttaa gcaataagcg aatataacac
     3001 ggtcgatcta aaaaggttag ctgggtgtgt tagaagccct gcaagacgac ctgcgcatta
     3061 ttactcacgg tacgacgcat tattactcac ggtacgaggc attcaccgcg tgccttacta
     3121 cgcgtattct taagcaataa gcgaatataa cacggtcgat ctaaaaaggt tagctgggtg
     3181 tgttagaagc cctgcaagac gacctgggca ttcaccgcgt gccttactac gcgtattctt
     3241 aagcaataag cgaatataac acggtcgatc taaaaaggtt agctgggtgt gttagaagcc
     3301 ctgcaagacg acctgggtac cgcattatta ctcacggtac gacgcattat tactcacggt
     3361 acgaggcatt caccgcgtgc cttactacgc gtattcttaa gcaataagcg aatataacac
     3421 ggtcgatcta aaaaggttag ctgggtgtgt tagaagccct gcaagacgac ctgggtacca
     3481 ggtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     3541 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattct
     3601 gcctcgagca gcgatatcat accgtcgaca atcaacctct ggattacaaa atttgtgaaa
     3661 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3721 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3781 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3841 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3901 tttccgggac tttcgctttc cccctcccta ttgccacggt ggaactcatc gccgcctgtc
     3961 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4021 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4081 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4141 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4201 tttgggccgc ctccccgcct gtctagagtc gacctgcaga agcttgcctc gagcagcgct
     4261 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg
     4321 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
     4381 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg
     4441 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca
     4501 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct
     4561 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt
     4621 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
     4681 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc
     4741 ttccctgtcc ttctgatttt gtaggtaacc acgtggtggt ggtgcggacc gagcggccgc
     4801 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4861 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4921 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     4981 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5041 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5101 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5161 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5221 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5281 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5341 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5401 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5461 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5521 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5581 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5641 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5701 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5761 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5821 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5881 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5941 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6001 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6061 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6121 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6181 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6241 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6301 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6361 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6421 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6481 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6541 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6601 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6661 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6721 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6781 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6841 ttcgttccac tgagcgtcag accccg
//
