LOCUS       sequence-385154-        7183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Astrocytic expression of ERT2-Cre-ERT2 recombinase 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 7183)
  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 7183)
  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-ERCreER-4x6T
FEATURES             Location/Qualifiers
     source          1..7183
                     /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      1528..1537
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1537..2472
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             2482..3504
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     D"
     CDS             3517..4452
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             3565..4458
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATA"
     polyA_signal    5026..5081
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   5118..5258
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5118..5247
                     /label=AAV2 ITR
     rep_origin      5333..5788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5420..5439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5630..5651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5805..5824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5924..5946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5984..6002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6070..6174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6175..6243,6244..7035)
                     /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 ttaatacctg gaattcccgg gtgagccgcc accatggctg gagacatgag agctgccaac
     1561 ctttggccaa gcccgctcat gatcaaacgc tctaagaaga acagcctggc cttgtccctg
     1621 acggccgacc agatggtcag tgccttgttg gatgctgagc cccccatact ctattccgag
     1681 tatgatccta ccagaccctt cagtgaagct tcgatgatgg gcttactgac caacctggca
     1741 gacagggagc tggttcacat gatcaactgg gcgaagaggg tgccaggctt tgtggatttg
     1801 accctccatg atcaggtcca ccttctagaa tgtgcctggc tagagatcct gatgattggt
     1861 ctcgtctggc gctccatgga gcacccagtg aagctactgt ttgctcctaa cttgctcttg
     1921 gacaggaacc agggaaaatg tgtagagggc atggtggaga tcttcgacat gctgctggct
     1981 acatcatctc ggttccgcat gatgaatctg cagggagagg agtttgtgtg cctcaaatct
     2041 attattttgc ttaattctgg agtgtacaca tttctgtcca gcaccctgaa gtctctggaa
     2101 gagaaggacc atatccaccg agtcctggac aagatcacag acactttgat ccacctgatg
     2161 gccaaggcag gcctgaccct gcagcagcag caccagcggc tggcccagct cctcctcatc
     2221 ctctcccaca tcaggcacat gagtaacaaa ggcatggagc atctgtacag catgaagtgc
     2281 aagaacgtgg tgcccctcta tgacctgctg ctggaggcgg cggacgccca ccgcctacat
     2341 gcgcccacta gccgtggagg ggcatccgtg gaggagacgg accaaagcca cttggccact
     2401 gcgggctcta cttcatcgca ttccttgcaa aagtattaca tcacggggga ggcagagggt
     2461 ttccctgcca cagctgtcga caatttactg accgtacacc aaaatttgcc tgcattaccg
     2521 gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag
     2581 gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg
     2641 tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt
     2701 ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta
     2761 aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg
     2821 gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta
     2881 gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc
     2941 caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc
     3001 gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc
     3061 catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg
     3121 ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat
     3181 aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag
     3241 ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct
     3301 aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc
     3361 gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg
     3421 accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg
     3481 gtgcgcctgc tggaagatgg cgatctcgag ccatctgctg gagacatgag agctgccaac
     3541 ctttggccaa gcccgctcat gatcaaacgc tctaagaaga acagcctggc cttgtccctg
     3601 acggccgacc agatggtcag tgccttgttg gatgctgagc cccccatact ctattccgag
     3661 tatgatccta ccagaccctt cagtgaagct tcgatgatgg gcttactgac caacctggca
     3721 gacagggagc tggttcacat gatcaactgg gcgaagaggg tgccaggctt tgtggatttg
     3781 accctccatg atcaggtcca ccttctagaa tgtgcctggc tagagatcct gatgattggt
     3841 ctcgtctggc gctccatgga gcacccagtg aagctactgt ttgctcctaa cttgctcttg
     3901 gacaggaacc agggaaaatg tgtagagggc atggtggaga tcttcgacat gctgctggct
     3961 acatcatctc ggttccgcat gatgaatctg cagggagagg agtttgtgtg cctcaaatct
     4021 attattttgc ttaattctgg agtgtacaca tttctgtcca gcaccctgaa gtctctggaa
     4081 gagaaggacc atatccaccg agtcctggac aagatcacag acactttgat ccacctgatg
     4141 gccaaggcag gcctgaccct gcagcagcag caccagcggc tggcccagct cctcctcatc
     4201 ctctcccaca tcaggcacat gagtaacaaa ggcatggagc atctgtacag catgaagtgc
     4261 aagaacgtgg tgcccctcta tgacctgctg ctggaggcgg cggacgccca ccgcctacat
     4321 gcgcccacta gccgtggagg ggcatccgtg gaggagacgg accaaagcca cttggccact
     4381 gcgggctcta cttcatcgca ttccttgcaa aagtattaca tcacggggga ggcagagggt
     4441 ttccctgcca cagcttgata gcgtgatgac atatgggcat tcaccgcgtg ccttactacg
     4501 cgtattctta agcaataagc gaatataaca cggtcgatct aaaaaggtta gctgggtgtg
     4561 ttagaagccc tgcaagacga cctgcgcatt attactcacg gtacgacgca ttattactca
     4621 cggtacgagg cattcaccgc gtgccttact acgcgtattc ttaagcaata agcgaatata
     4681 acacggtcga tctaaaaagg ttagctgggt gtgttagaag ccctgcaaga cgacctgggc
     4741 attcaccgcg tgccttacta cgcgtattct taagcaataa gcgaatataa cacggtcgat
     4801 ctaaaaaggt tagctgggtg tgttagaagc cctgcaagac gacctgggta ccgcattatt
     4861 actcacggta cgacgcatta ttactcacgg tacgaggcat tcaccgcgtg ccttactacg
     4921 cgtattctta agcaataagc gaatataaca cggtcgatct aaaaaggtta gctgggtgtg
     4981 ttagaagccc tgcaagacga cctgggtacc aggtaaccgc ggtgaaataa aggaaattta
     5041 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc agtgcacgtg gtgtggtggt
     5101 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     5161 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     5221 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5281 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5341 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5401 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5461 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5521 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5581 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5641 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5701 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5761 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5821 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5881 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5941 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6001 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6061 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6121 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6181 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6241 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6301 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6361 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6421 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6481 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6541 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6601 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6661 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6721 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6781 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6841 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6901 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6961 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7021 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccg
//
