LOCUS       sequence-497742-        6795 bp    DNA     circular SYN 16-JAN-2026
DEFINITION  Cre-dependent AAV expressing mCherry and an shRNA targeting Numb 
            under EF1-alpha promoter for selective Numb knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6795)
  AUTHORS   Shin J, Oh H, Park JH, Bae H, Yang CM, Lee M, Kim S, Heo WD
  TITLE     Dysregulation of FGFR1 signaling in the hippocampus facilitates 
            depressive disorder.
  JOURNAL   Exp Mol Med. 2025 Aug;57(8):1818-1836. doi: 
            10.1038/s12276-025-01519-9. Epub 2025 Aug 15.
  PUBMED    40813470
REFERENCE   2  (bases 1 to 6795)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 16, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s12276-025-01519-9"; journalName: "Exp Mol Med"; date: 
            "2025-08"; volume: "57"; issue: "8"; pages: "1818-1836"
COMMENT     Sequence Label: AAV-EF1-alpha-DIO-DSE-mCherry-PSE-shRNA-Numb
FEATURES             Location/Qualifiers
     source          1..6795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1446..1479)
                     /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(1530..1563)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1652..1672)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1772..2482)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2050..2069)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2319..2337
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2479..2488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2610..2643
                     /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    2694..2727
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2763..2782
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2922..3510
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2975..2995)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3393..3404)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3542..4018
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3741..3760)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4058..4198
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4058..4187
                     /label=AAV2 ITR
     rep_origin      4273..4728
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4360..4379)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4570..4591
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4745..4764)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4864..4886
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4924..4942)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5010..5114
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5115..5183,5184..5975)
                     /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(5333..5352)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6146..6734
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6635..6654
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagattc gctagcggat
     1441 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
     1501 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
     1561 tattattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
     1621 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcttcctgcc
     1681 cgaccttggc gcgcgctcgg cgcgcggtca cgctccgtca cgtggtgcgt tttgcctgcg
     1741 cgtctttcca ctgggttaat taagcggccg cttacttgta cagctcgtcc atgccgccgg
     1801 tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg
     1861 aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg
     1921 ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct
     1981 gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc
     2041 agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct
     2101 tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc
     2161 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca
     2221 agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag
     2281 gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg
     2341 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
     2401 tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat
     2461 cctcctcgcc cttgctcacc atggtggcgg atgctaccgg tggatcccgg gcccgcggta
     2521 ccgtcgactg cagctcctga tcggcctctt cattcttctc aaccggttta gttcttctga
     2581 tgcagcacag catggggcgg cgcgccgcga taacttcgta taaagtatcc tatacgaagt
     2641 tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt
     2701 cgtataatgt atgctatacg aagttatgtt tatctgcagt tttaaaatta tgttttaaaa
     2761 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttgggt ttatatatct
     2821 tgtggaaagg acgcggtttc ctaggtctgt gaagatgccg taaacctgac ccatttacgg
     2881 catcttcaca gatttttgaa ttcgatatca agcttatcga taatcaacct ctggattaca
     2941 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3001 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3061 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3121 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3181 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3241 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3301 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     3361 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3421 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3481 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     3541 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     3601 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     3661 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     3721 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     3781 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     3841 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     3901 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     3961 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     4021 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     4081 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4141 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4201 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4261 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4321 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     4381 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4441 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4501 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4561 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     4621 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     4681 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4741 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4801 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4861 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4921 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4981 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5041 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5101 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5161 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5221 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5281 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5341 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5401 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5461 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5521 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5581 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5641 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5701 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5761 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5821 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5881 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5941 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6001 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6061 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6121 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6181 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6241 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6301 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6361 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6421 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6481 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6541 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6601 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6661 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6721 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6781 cttttgctca catgt
//
