LOCUS       sequence-489351-        7843 bp    DNA     circular SYN 15-OCT-2025
DEFINITION  Floxed (DIO) AAV transgene designed to be packaged in AAV and to 
            express the amyloid precursor protein (App) with familial mutations 
            AppNL-G-F in a Cre-recombinase dependent manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7843)
  TITLE     AAV transgenes to express familial Alzheimer's disease
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7843)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hsyn-DIO-AppNL-G-F-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7843
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(786..1499,1500..1502,1503..1505))
                     /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             complement(join(789..1499,1500..1502,1503..1505))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(831..852)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1180..1199
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1439..1460
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1506..1562)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    3828..3861
                     /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    3912..3945
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3970..4558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4023..4043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4441..4452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4590..5066
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4789..4808)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5106..5246
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5106..5235
                     /label=AAV2 ITR
     rep_origin      5321..5776
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5408..5427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5618..5639
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5793..5812)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5912..5934
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5972..5990)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6058..6162
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6163..6231,6232..7023)
                     /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(6381..6400)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7194..7782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7683..7702
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
      841 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
      901 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
      961 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1021 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1081 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1141 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1201 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1261 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     1321 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     1381 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     1441 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     1501 accattggcc cgggattctc ttcgacatcc cctgcttgtt tcaacaggga gaagttagtg
     1561 gcaccggtgt tctgcatttg ctcaaagaac ttgtaagttg gattctcata tccgttctgc
     1621 tgcatcttgg agagatggcg ctcctctggg gtcacggcgg cgtcgacctc caccacgcca
     1681 tgatggatgg atgtgtactg tttcttcttc aacatcacca gggtgatgac aaacacggtt
     1741 gctatgacaa cgccgcccac catgagtccg atgatggcgc ctttgttcga acccacatct
     1801 ccagcaaaga acaccagttt ttgatggtgg acttcatatc ctgaatcatg tctgaattct
     1861 gcatctagat tcacttccga gatctcttcc gtcttgatgt ttgtcagccc agaacctggt
     1921 cgagtggtca gtcctcggtc agcagcgggg cgggcgtcaa caggctcgac ttcattttcg
     1981 gtattggctg gcacagagtc caccccaaaa gggtgccacg gctggaggtc atccaggctg
     2041 aattccccat tcacgggaag gagctccacg gtggtcttgg tttccgtcag cgaaggcatg
     2101 agagcgtcgt ttccgtagct gattctgggc tcactgatca tgttggccaa gacatcgtcg
     2161 gagtagttct gctccttctg aagcagctca tcgacttcat cttgaatctc ctcagccacc
     2221 gcagggacat tgtagagcag ggacagagac tggttcatgc gctcgtagat cacacggagg
     2281 tgtgtcataa cctgggaccg gatctgagca gctttcttgg ggtccaccat gcgcacatgt
     2341 tcaaaatgct ttagggtgtg ctgtctgtct ttctgctccg cacggacgta cttcttcagc
     2401 atgttgaaca catgatgagg ccttgggggc accgcctgca gtgcagtgat gtaattctcg
     2461 agggccaggc ggcggcggtc attgagcatg gcttcaactc tggccatgtg tgtctctaca
     2521 agctgctgtc tctcattggc tgcttcctgt tccagagatt ccactttctc ctggaaatgc
     2581 tggataacgg ccttcttgtc agctttgggc aagttcttgg cttgacgctc tgcctcttcc
     2641 cattctctca tgacctggga cattctctct cggtgcttgg cttccagcct ctctttggct
     2701 ttctggaaat gggcatgctc gttctcgtcc ccgggtgtct ccaggtactt gtcgacggcg
     2761 tcgggggtgc tggctgctgt cgtgggaaac acgctgccac acaccgccat gcagtactct
     2821 tccgtgtcaa agttgttcct gttgccgcca catccgccgt aaaagaatgg gacacacttc
     2881 ccttcagtga catcaaagta ccagcgggag atcattgcgc ggcatggccc ggtctcggct
     2941 tgttcagagc acacctctcg gaccacctcc tccacggact cagtggtggt tgtggtggtg
     3001 gtggcagtgc tggttgttct ctcggtggcc tcttcgtagg gctcctcggc ctcctcctcc
     3061 acctcgtccc catcctccac atcctcatca tcatcagctt cctcttcctc aacatcagcc
     3121 acttcctcct cttcggcgac ttctactact ttgtcttcac cgccatcagc gtagtctgtg
     3181 tccgctccac cccaccagac atcagagtca tcctcctctg catccgcaga atccacgctg
     3241 tcgctttcct cggccaacgg gcagcataca aactctaccc ctcggaactt gtcgatgccg
     3301 cagggcagca gcatgccata gtcgtgcaag ttagtgctct tctcgctgca tgtctctttg
     3361 gcgacggtgt gccagtgaag atgggtctca caaacatcca tccgctcctg gtgtaggaac
     3421 ttgcacttgt cgggcacgag aagggcgtcg ctcacaaact caccaactag gcaacggtaa
     3481 ggaatcacga tgtgggtgtg tgtcttgcac tgcttgcggc cccgcttgca ccagttctgg
     3541 atggtcactg gctggttggc ttccaccacg tttgtgatct gcagttcagg gtagacctct
     3601 tggcagtact gcaagatgcc ctccttggtg ccaatgcagg ttttggtccc tgacgggtct
     3661 gactcccact ttccattctg cacattcatg tgcatgttga gtttaccaca gaacatggcg
     3721 atctggggtt ctgccagcag cccggcgttg ccatcagtgg gtacctccag agcccgaacc
     3781 gtccaggcgg ccagcaggag cagtgccaag ctgggcagca tgctagcata acttcgtata
     3841 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
     3901 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
     3961 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4021 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4081 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4141 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     4201 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     4261 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4321 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     4381 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     4441 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     4501 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     4561 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     4621 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     4681 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     4741 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     4801 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     4861 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     4921 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     4981 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     5041 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     5101 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     5161 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     5221 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     5281 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     5341 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     5401 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5461 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5521 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5581 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5641 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5701 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5761 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5821 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5881 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5941 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     6001 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6061 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6121 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6181 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6241 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6301 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6361 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6421 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6481 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6541 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6601 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6661 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6721 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6781 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6841 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6901 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6961 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7021 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7081 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7141 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7201 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7261 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7321 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7381 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7441 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7501 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7561 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7621 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7681 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7741 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7801 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
