LOCUS       sequence-385111-        6644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Astrocytic expression of membrane-targeted FLAG 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 6644)
  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 6644)
  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-smFLAG-4x6T-WPRE
FEATURES             Location/Qualifiers
     source          1..6644
                     /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..1695
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1699..1722
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1726..1749
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2278..2301
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2308..2331
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2344..2367
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2374..2397
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2608..2631
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2635..2658
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2662..2685
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    3408..3996
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3461..3481)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3879..3890)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4054..4530
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4253..4272)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4579..4719
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4579..4708
                     /label=AAV2 ITR
     rep_origin      4794..5249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4881..4900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5091..5112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5266..5285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5385..5407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5445..5463)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5531..5635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5636..5704,5705..6496)
                     /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 ggactacaag
     1681 gacgacgacg ataaaggaga ttacaaagat gacgatgata agggcgatta taaggacgac
     1741 gatgacaagg gcggcgtgtc gaaaggcgag gagctgttca caggcgtggt ccccatcctg
     1801 gttgagctgg acggtgacgt taacggccac aaatttagcg tacgtggtga gggcgagggt
     1861 gacgctacca acggaaagct gaccctgaaa ttcatctgca caactggcaa gctgccggtg
     1921 ccctggccca cgctggtaac cactctgggc ggaggcgttc aatgtttcag tcgttacccg
     1981 gatcacatga agcagcatga cttcttcaag tcggccatgc ccgaaggcta cgtgcaggag
     2041 cgcaccatca gttttaagga tgacggcacc tacaagactc gcgccgaggt caagttcgaa
     2101 ggagacaccc tggttaatcg tatcgagctc aaaggcatcg atttcaagga ggacggaaat
     2161 attctgggcc acaagctgga gtacaatttt aacagccaca atgtttacat tactgccgac
     2221 aagcagaaga acggcatcaa ggctaatttc aagattcgtc acaatgtgga gggaggcgac
     2281 tataaggatg acgatgacaa acaacaagac tataaggacg atgatgacaa aggtcagcaa
     2341 ggcgattaca aggacgatga cgataagcag caagattaca aggatgatga tgataaggga
     2401 ggcgacggaa gcgtgcagct ggcagatcat taccaacaga acacgcccat tggtgacggt
     2461 ccagtcctgc tgcctgataa ccactacttg tccacccagt cggtgctgtc gaaggacccg
     2521 aacgagaagc gtgaccacat ggtgctgctg gagttcgtga ccgccgcagg aatcaccctc
     2581 ggcatggatg agctttacaa gggcggagat tataaagatg acgacgacaa gggcgactac
     2641 aaagatgatg atgacaaggg tgactacaaa gacgacgatg ataaataaaa taggtaaagc
     2701 ggccgcgact ctagatcata tgggcattca ccgcgtgcct tactacgcgt attcttaagc
     2761 aataagcgaa tataacacgg tcgatctaaa aaggttagct gggtgtgtta gaagccctgc
     2821 aagacgacct gcgcattatt actcacggta cgacgcatta ttactcacgg tacgaggcat
     2881 tcaccgcgtg ccttactacg cgtattctta agcaataagc gaatataaca cggtcgatct
     2941 aaaaaggtta gctgggtgtg ttagaagccc tgcaagacga cctgggcatt caccgcgtgc
     3001 cttactacgc gtattcttaa gcaataagcg aatataacac ggtcgatcta aaaaggttag
     3061 ctgggtgtgt tagaagccct gcaagacgac ctgggtaccg cattattact cacggtacga
     3121 cgcattatta ctcacggtac gaggcattca ccgcgtgcct tactacgcgt attcttaagc
     3181 aataagcgaa tataacacgg tcgatctaaa aaggttagct gggtgtgtta gaagccctgc
     3241 aagacgacct gggtaccagg taaagcggcc gcgactctag atcataatca gccataccac
     3301 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     3361 taaaatgaat gcaattctgc ctcgagcagc gatatcatac cgtcgacaat caacctctgg
     3421 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3481 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3541 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3601 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3661 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggtgg
     3721 aactcatcgc cgcctgtctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3781 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     3841 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3901 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3961 agacgagtcg gatctccctt tgggccgcct ccccgcctgt ctagagtcga cctgcagaag
     4021 cttgcctcga gcagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca
     4081 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     4141 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     4201 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     4261 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     4321 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     4381 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     4441 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     4501 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtggtggtgg
     4561 tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4621 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4681 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4741 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4801 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4861 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4921 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4981 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     5041 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5101 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     5161 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5221 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5281 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5341 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5401 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5461 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5521 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5581 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5641 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5701 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5761 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5821 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5881 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5941 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6001 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6061 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6121 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6181 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6241 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6301 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6361 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6421 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6481 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6541 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6601 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccg
//
