LOCUS       sequence-385571-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent AAV expression of membrane-targeted V5 spaghetti 
            monster reporter preferentially in astrocytes; astrocyte selectivity
            generated with 4x6T miRNA targeting cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  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 7019)
  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-CAG-flex-lck-smV5-4x6T
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1162..1617)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1320)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1511..1530
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   1689..1818
                     /label=AAV2 ITR
     promoter        2129..2406
                     /label=chicken beta-actin promoter
     primer_bind     2685..2707
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2769..2788
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2823..2856
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2919..2952
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(3558..3599)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3609..3650)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3660..3701)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3915..3956)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3966..4007)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4026..4067)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4077..4118)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4650..4691)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4701..4742)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(4752..4793)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     regulatory      4793..4802
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4934..4943
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4972..5005)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(5076..5109)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5151..5739
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5204..5224)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5622..5633)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5782..5903
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5793..5812)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5847..5866
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6065..6194
                     /label=AAV2 ITR
     rep_origin      complement(6431..7019)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6511..6530)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1201 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     1261 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     1321 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     1381 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     1441 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     1501 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     1561 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     1621 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     1681 tacgccagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg
     1741 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     1801 ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc
     1861 catgctctag gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca
     1921 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1981 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     2041 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     2101 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     2161 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     2221 gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gsggggsggg gsggggsgag
     2281 gggsggggsg gggsgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     2341 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     2401 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     2461 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     2521 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     2581 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg
     2641 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     2701 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     2761 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc
     2821 ccataacttc gtataaagta tcctatacga agttatatca aaataggaag accaatgctt
     2881 caccatcgac ccgaattgcc aagcatcacc atcgacccat aacttcgtat aatgtatgct
     2941 atacgaagtt atactaggga tccccccgtc gcggccgctt tacctggtac ccaggtcgtc
     3001 ttgcagggct tctaacacac ccagctaacc tttttagatc gaccgtgtta tattcgctta
     3061 ttgcttaaga atacgcgtag taaggcacgc ggtgaatgcc tcgtaccgtg agtaataatg
     3121 cgtcgtaccg tgagtaataa tgcggtaccc aggtcgtctt gcagggcttc taacacaccc
     3181 agctaacctt tttagatcga ccgtgttata ttcgcttatt gcttaagaat acgcgtagta
     3241 aggcacgcgg tgaatgccca ggtcgtcttg cagggcttct aacacaccca gctaaccttt
     3301 ttagatcgac cgtgttatat tcgcttattg cttaagaata cgcgtagtaa ggcacgcggt
     3361 gaatgcctcg taccgtgagt aataatgcgt cgtaccgtga gtaataatgc gcaggtcgtc
     3421 ttgcagggct tctaacacac ccagctaacc tttttagatc gaccgtgtta tattcgctta
     3481 ttgcttaaga atacgcgtag taaggcacgc ggtgaatgcc catatgctaa tcggtcgcgg
     3541 ccgctttacc tattttaggt actatccagt cccagcaacg ggttcgggat gggcttgccc
     3601 tgctgttgcg tactgtcgag gcccaacagt ggatttggta ttggctttcc ttgttgctga
     3661 gtgctgtcga ggcccagcaa gggatttggt atgggcttgc ctccttggcc cttgtacagc
     3721 tcgtccattc ccaacgtaat gccggcggcg gtcacgaact ccaacaacac catgtgatct
     3781 cgtttttcgt ttgggtcctt actcagaacg ctctgagtcg acagatagtg attatccggc
     3841 aacagcacgg ggccatctcc gatcggcgta ttctgctgat agtggtccgc cagttgaaca
     3901 cttccatcgc cacctgtcga gtcgaggccc agcagagggt tggggattgg cttgccttgc
     3961 tgctgggtgc tatccaggcc gagcagggga ttgggtatcg gtttgccacc gccttgctgg
     4021 cctccggtgc tgtcgagtcc cagcaagggg ttagggatgg gctttccctg ttgctgcgtg
     4081 ctatccaagc ccagcagggg attcgggatc ggcttgccac caccttccac gttgtgccga
     4141 attttgaaat tcgcctttat gccgttcttc tgcttgtcgg cagtaatgta gacgttatgg
     4201 ctgttaaagt tgtattccag cttgtgaccc agaatattac cgtcttcctt gaaatcaatt
     4261 cccttaagtt ctatcctgtt caccaaggtg tctccctcga acttcacctc ggctcgcgtc
     4321 ttgtaagtac cgtcatcttt gaaactgatc gtccgttcct gcacgtatcc ctccggcata
     4381 gcactcttaa agaagtcatg ctgcttcatg tggtctgggt aacggctgaa acactggact
     4441 ccgccaccca gggttgtaac caaggtcggc cacggcaccg gcagctttcc cgtggtgcat
     4501 ataaacttca gcgtcaattt tccgttggtg gcatctccct ctccttctcc gcgcacacta
     4561 aacttgtgtc cgttaacgtc accgtcgagc tcaaccagaa ttgggaccac gccggtaaac
     4621 agttcttcgc ccttactcac accttggccc gtggagtcca ggcccaacag agggttaggg
     4681 ataggcttac cttgttgctg tgtgctatcc agaccgagaa gtgggtttgg aatgggctta
     4741 ccctgctgct gagtactatc aagtccaagg agggggttcg gtataggttt tcccatggtg
     4801 gcgaccggtg gatcccgggc ccgcggtacc gtcgactgca gaattcgaag cttcggcggg
     4861 ggataatggc agttctcaca cacgtcaatg ttctccatcc agtcatcttc agggtttgag
     4921 ctgcagccac agcccatggt ggcagatctt acgcgtgtac cgaattctag tataacttcg
     4981 tataggatac tttatacgaa gttatcattg ggattcttcc tattttgatc caagcatcac
     5041 catcgaccct ctagtccaga tctcaccatc gacccataac ttcgtatagc atacattata
     5101 cgaagttatg tccctcgaag aggttcgaat tcgatatcaa gcttatcgat aatcaacctc
     5161 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     5221 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     5281 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     5341 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     5401 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     5461 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     5521 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     5581 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     5641 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     5701 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacccgggc
     5761 ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     5821 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     5881 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     5941 agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg
     6001 ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac
     6061 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     6121 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     6181 gagcgagcgc gcagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     6241 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     6301 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6361 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6421 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6481 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6541 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6601 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6661 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6721 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6781 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6841 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6901 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6961 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
