LOCUS       sequence-440798-        7246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mir30 based shRNA strategy, control shRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7246)
  AUTHORS   Murdock MH, Yang CY, Sun N, Pao PC, Blanco-Duque C, Kahn MC, Kim T, 
            Lavoie NS, Victor MB, Islam MR, Galiana F, Leary N, Wang S, Bubnys 
            A, Ma E, Akay LA, Sneve M, Qian Y, Lai C, McCarthy MM, Kopell N, 
            Kellis M, Piatkevich KD, Boyden ES, Tsai LH
  TITLE     Multisensory gamma stimulation promotes glymphatic clearance of 
            amyloid.
  JOURNAL   Nature. 2024 Mar;627(8002):149-156. doi: 10.1038/s41586-024-07132-6.
            Epub 2024 Feb 28.
  PUBMED    38418876
REFERENCE   2  (bases 1 to 7246)
  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"; doi: 
            "10.1038/s41586-024-07132-6"; journalName: "Nature"; date: 
            "2024-03"; volume: "627"; issue: "8002"; pages: "149-156"
COMMENT     Sequence Label: pAAV-GFAP-eGFP-shLacZ
FEATURES             Location/Qualifiers
     source          1..7246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   45..174
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     misc_feature    190..307
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     regulatory      2564..2573
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2570..2572,2573..2575,2576..3289)
                     /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(2570..2572,2573..2575,2576..3286)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2615..2636)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2876..2895)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3223..3244
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           3350..3364
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    3417..4005
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3470..3490)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3888..3899)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4037..4513
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4236..4255)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4553..4693
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4553..4682
                     /label=AAV2 ITR
     rep_origin      4768..5223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4855..4874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5065..5086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5240..5259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5359..5381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5419..5437)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5505..5609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5610..5678,5679..6470)
                     /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"
     rep_origin      6641..7229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7130..7149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
      121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
      181 gcacgcgttt tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
      241 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
      301 aaattttcgg gtttattaca gggacagcag agatccagtt tggttaatta acccacctcc
      361 ctctctgtgc tgggactcac agagggagac ctcaggaggc agtctgtcca tcacatgtcc
      421 aaatgcagag cataccctgg gctgggcgca gtggcgcaca actgtaattc cagcactttg
      481 ggaggctgat gtggaaggat cacttgagcc cagaagttct agaccagcct gggcaacatg
      541 gcaagaccct atctctacaa aaaaagttaa aaaatcagcc acgtgtggtg acacacacct
      601 gtagtcccag ctattcagga ggctgaggtg aggggatcac ttaaggctgg gaggttgagg
      661 ctgcagtgag tcgtggttgc gccactgcac tccagcctgg gcaacagtga gaccctgtct
      721 caaaagacaa aaaaaaaaaa aaaaaaaaaa agaacatatc ctggtgtgga gtaggggacg
      781 ctgctctgac agaggctcgg gggcctgagc tggctctgtg agctggggag gaggcagaca
      841 gccaggcctt gtctgcaagc agacctggca gcattgggct ggccgccccc cagggcctcc
      901 tcttcatgcc cagtgaatga ctcaccttgg cacagacaca atgttcgggg tgggcacagt
      961 gcctgcttcc cgccgcaccc cagcccccct caaatgcctt ccgagaagcc cattgagcag
     1021 ggggcttgca ttgcacccca gcctgacagc ctggcatctt gggataaaag cagcacagcc
     1081 ccctaggggc tgcccttgct gtgtggcgcc accggcggtg gagaacaagg ctctattcag
     1141 cctgtgccca ggaaagggga tcaggggatg cccaggcatg gacagtgggt ggcagggggg
     1201 gagaggaggg ctgtctgctt cccagaagtc caaggacaca aatgggtgag gggactgggc
     1261 agggttctga ccctgtggga ccagagtgga gggcgtagat ggacctgaag tctccaggga
     1321 caacagggcc caggtctcag gctcctagtt gggcccagtg gctccagcgt ttccaaaccc
     1381 atccatcccc agaggttctt cccatctctc caggctgatg tgtgggaact cgaggaaata
     1441 aatctccagt gggagacgga ggggtggcca gggaaacggg gcgctgcagg aataaagacg
     1501 agccagcaca gccagctcat gtgtaacggc tttgtggagc tgtcaaggcc tggtctctgg
     1561 gagagaggca cagggaggcc agacaaggaa ggggtgacct ggagggacag atccaggggc
     1621 taaagtcctg ataaggcaag agagtgccgg ccccctcttg ccctatcagg acctccactg
     1681 ccacatagag gccatgattg acccttagac aaagggctgg tgtccaatcc cagcccccag
     1741 ccccagaact ccagggaatg aatgggcaga gagcaggaat gtgggacatc tgtgttcaag
     1801 ggaaggactc caggagtctg ctgggaatga ggcctagtag gaaatgaggt ggcccttgag
     1861 ggtacagaac aggttcattc ttcgccaaat tcccagcacc ttgcaggcac ttacagctga
     1921 gtgagataat gcctgggtta tgaaatcaaa aagttggaaa gcaggtcaga ggtcatctgg
     1981 tacagccctt ccttcccttt tttttttttt ttttttgtga gacaaggtct ctctctgttg
     2041 cccaggctgg agtggcgcaa acacagctca ctgcagcctc aacctactgg gctcaagcaa
     2101 tcctccagcc tcagcctccc aaagtgctgg gattacaagc atgagccacc ccactcagcc
     2161 ctttccttcc tttttaattg atgcataata attgtaagta ttcatcatgg tccaaccaac
     2221 cctttcttga cccaccttcc tagagagagg gtcctcttgc ttcagcggtc agggccccag
     2281 acccatggtc tggctccagg taccacctgc ctcatgcagg agttggcgtg cccaggaagc
     2341 tctgcctctg ggcacagtga cctcagtggg gtgaggggag ctctccccat agctgggctg
     2401 cggcccaacc ccaccccctc aggctatgcc agggggtgtt gccaggggca cccgggcatc
     2461 gccagtctag cccactcctt cataaagccc tcgcatccca ggagcgagca gagccagagc
     2521 aggttggaga ggagacgcat cacctccgct gctcgccggg atggccgcca tggtgagcaa
     2581 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     2641 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     2701 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     2761 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2821 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2881 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2941 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3001 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     3061 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3121 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3181 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3241 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat aagctaggaa
     3301 ggtatattgc tgttgacagt gagcgtcgct aaatactggc aggcgtttag tgaagccaca
     3361 gatgtaaacg cctgccagta tttagcgatg cctactgcct cggctagctt atcgataatc
     3421 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3481 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3541 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3601 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3661 ggggcattgc caccacctgt cagctccttt ccggaacttt cgctttcccc ctccctattg
     3721 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3781 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3841 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3901 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3961 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     4021 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     4081 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     4141 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     4201 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     4261 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     4321 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     4381 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     4441 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     4501 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4561 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4621 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4681 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4741 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4801 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4861 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4921 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4981 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5041 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5101 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5161 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5221 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5281 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5341 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5401 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5461 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5521 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5581 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5641 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5701 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5761 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5821 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5881 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5941 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6001 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6061 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6121 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6181 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6241 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6301 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6361 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6421 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6481 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6541 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6601 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6661 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6721 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6781 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6841 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6901 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6961 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7021 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7081 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7141 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7201 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggc
//
