LOCUS       sequence-467452-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Improved microglia-targeted transgene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  AUTHORS   Aoki R, Konno A, Hosoi N, Kawabata H, Hirai H
  TITLE     AAV vectors for specific and efficient gene expression in microglia.
  JOURNAL   Cell Rep Methods. 2025 Jul 25:101116. doi: 
            10.1016/j.crmeth.2025.101116.
  PUBMED    40744011
REFERENCE   2  (bases 1 to 7455)
  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: "Cell Rep 
            Methods. 2025 Jul 25:101116. doi: 10.1016/j.crmeth.2025.101116."
COMMENT     Sequence Label: 
            pAAV/mIba1.GFP.miR-9.T.miR-129-2-3p.T.WPRE.miR-9.T.miR-129-2-3p.T.SV
            40pA
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      444..1032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     933..952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      2994..3003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3000..3002,3003..3005,3006..3719)
                     /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(3000..3002,3003..3005,3006..3716)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3045..3066)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3306..3325)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3653..3674
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3951..4539
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4004..4024)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4422..4433)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4834..4853)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4888..4907
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5003..5132
                     /label=AAV2 ITR
     rep_origin      5503..6016
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5590..5609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5800..5821
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6498..6517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6617..6639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6677..6695)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6763..6867
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6868..6936,6937..7455,1..273)
                     /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     complement(7086..7105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
       61 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
      121 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
      181 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
      241 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
      301 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
      361 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      421 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      481 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      541 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      601 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      661 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      721 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      781 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      841 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      901 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      961 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     1021 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     1081 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     1141 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     1201 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt gtagttaatg
     1261 attaacccgc catgctactt atctacgtag ccatgctcta gactcgagta ctataggatg
     1321 catcgtgaaa acctcgtttc caccaggaac tgaggttgct gctggaggaa atctcccatg
     1381 aagagctgtt aactggggaa ctggttggat ccagctccta caagctctcc tttaatttcc
     1441 atacacatgc caagctgtga acacctgcac acacagtaca aataaaacag tagaaatgaa
     1501 atgaaaatta aaacaaaaac agcatacttt ctcctttcta cccacatatc tcctgcatcc
     1561 ctgagagcaa tttcttggcc atcttccata atcatgctat tcaaacctgc cctctaagta
     1621 cagagctctg aatggagacc acgggagcag acttttcatc tgttggctcc ctgggcctag
     1681 cagtgtaccc tgcaaacagg aaaccttcgc tcatcattta tcacatgaat gtggctagag
     1741 aactgtcccc accagacact gagagcctct cacagggcca gagggtggct gcctcaaagg
     1801 caggcgagag gctgcagtct tctgcagagt ggacagatac tgcctgccat acaaggggac
     1861 atagcctggg gcggggggca caagggaagg ggcctgagtg cattggagtc acagccctcc
     1921 ctcccctggt gacccagtga ctgaggggac caaggctatc cctggtatga ggggagggca
     1981 tttgcatgag ttagacccct gacagcccat ggtggggaca ggaagtagct ctgtctactg
     2041 tctctttcag tctcactttc tgttcctcca aagtgtctct tcaaactctc aaatgacttt
     2101 gtagattcag cctaagatct caaagcaggt gaggcaggag gctggcactc aagagcctga
     2161 cgaacacaaa cccaagtcct tctgggacat gtctccatgt gtccccagtg ctcctctgta
     2221 gcccctgaga gaggtttctt tcctttccag ggcgctaggc tcagctcacc ccattcctgg
     2281 agcagcctgc agacttcatc ctctctcttc catcccgggg aaagtcagcc agtcctcctc
     2341 agctgcctgt cttaacctgc atcatgaagc ctgaggagat ttcaagtaaa ccctcccaag
     2401 ccccacctca ggatctgggg aaagccactg tctaccgcat ccttggtttg agacaggttc
     2461 ttactgtgtt ggcctggctg ggttccaact cccaggatcc tctagcctca gcctctctta
     2521 ggtgctagat tgcaggccta gggtatcacg ctcggtccct cagatcctgg tgcctttcca
     2581 cacctccggg agctgatcta agtctttctc ccacgtacag tctgcttctt tggaggagct
     2641 gtgtggagct aggtgtgtta cagggctgta gctccagcca tctgggaggc tgaggcagga
     2701 tggctacttc aattctcaag agcctggata ggagaccctg cccccacccc acccccaaag
     2761 taatttttca gcacattact tcttcatctc ctctctcaac ccgttctgcc ttctcctggg
     2821 gtgctggtgt cagcagaagc tgatgtggaa gtgatgcctg ggagttagca agggaatgag
     2881 tggaaagggg aagtgtgaga acggtcccag aagagactgg ggagctggtg gagagaggac
     2941 ccagcggaca gactgccagc ctaagacaac cagcgtctga ggagccaccg gtcgccacca
     3001 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3061 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3121 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3181 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     3241 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3301 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3361 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     3421 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     3481 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     3541 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     3601 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     3661 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     3721 gcggccgctc atacagctag ataaccaaag cgattcatac agctagataa ccaaagcaat
     3781 tgtcatacag ctagataacc aaagtcactc atacagctag ataaccaaag gctagcatgc
     3841 tttttggggt aagggcttcg atatgctttt tggggtaagg gcttcttaag atgctttttg
     3901 gggtaagggc tttcacatgc tttttggggt aagggcttgg ccgcatcgat aatcaacctc
     3961 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     4021 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     4081 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     4141 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     4201 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     4261 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     4321 acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg
     4381 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     4441 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4501 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgatgccgt atacggtacc
     4561 tcatacagct agataaccaa agcgattcat acagctagat aaccaaagca attgtcatac
     4621 agctagataa ccaaagtcac tcatacagct agataaccaa aggctagcat gctttttggg
     4681 gtaagggctt cgatatgctt tttggggtaa gggcttctta agatgctttt tggggtaagg
     4741 gctttcacat gctttttggg gtaagggctt ggatcctcta gtagagtcga ggaattcact
     4801 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4861 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4921 atgtctgtcg acccccaacg cgttggagct ctagagcatg gctacgtaga taagtagcat
     4981 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     5041 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     5101 cgggcggcct cagtgagcga gcgagcgcgc agctggcgta atagcgaaga ggcccgcacc
     5161 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgattccg ttgcaatggc
     5221 tggcggtaat attgttctgg atattaccag caaggccgat agtttgagtt cttctactca
     5281 ggcaagtgat gttattacta atcaaagaag tattgcgaca acggttaatt tgcgtgatgg
     5341 acagactctt ttactcggtg gcctcactga ttataaaaac acttctcagg attctggcgt
     5401 accgttcctg tctaaaatcc ctttaatcgg cctcctgttt agctcccgct ctgattctaa
     5461 cgaggaaagc acgttatacg tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5521 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5581 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5641 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5701 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5761 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5821 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5881 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5941 tattaacgct tacaatttaa atatttgctt atacaatctt cctgtttttg gggcttttct
     6001 gattatcaac cggggtacat atgattgaca tgctagtttt acgattaccg ttcatcgatt
     6061 ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa
     6121 aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg
     6181 tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg
     6241 cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc
     6301 ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg
     6361 ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga
     6421 tgttggaatc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     6481 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     6541 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6601 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6661 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     6721 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6781 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6841 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6901 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6961 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     7021 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     7081 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     7141 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     7201 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     7261 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     7321 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     7381 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     7441 ttaactggcg aacta
//
