LOCUS       sequence-489352-        7846 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 7846)
  TITLE     AAV transgenes to express familial Alzheimer's disease
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7846)
  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            "Unpublished"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-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7846
                     /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(786..1496)
                     /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(1064..1083)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1333..1351
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1512..1565)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    3831..3864
                     /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    3915..3948
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3973..4561
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4026..4046)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4444..4455)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4593..5069
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4792..4811)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5109..5249
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5109..5238
                     /label=AAV2 ITR
     rep_origin      5324..5779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5411..5430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5621..5642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5796..5815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5915..5937
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5975..5993)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6061..6165
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6166..6234,6235..7026)
                     /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(6384..6403)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7197..7785
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7686..7705
                     /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 ccggtggagt ggcggccctc ggcgcgttcg
      841 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
      901 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
      961 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     1021 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     1081 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     1141 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     1201 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     1261 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     1321 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     1381 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     1441 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
     1501 gatcccggtg cagggccggg attctcctcc acgtcaccgc atgttagaag acttcctctg
     1561 ccctcaccgg tgttctgcat ttgctcaaag aacttgtaag ttggattctc atatccgttc
     1621 tgctgcatct tggagagatg gcgctcctct ggggtcacgg cggcgtcgac ctccaccacg
     1681 ccatgatgga tggatgtgta ctgtttcttc ttcaacatca ccagggtgat gacaaacacg
     1741 gttgctatga caacgccgcc caccatgagt ccgatgatgg cgcctttgtt cgaacccaca
     1801 tctccagcaa agaacaccag tttttgatgg tggacttcat atcctgaatc atgtctgaat
     1861 tctgcatcta gattcacttc cgagatctct tccgtcttga tgtttgtcag cccagaacct
     1921 ggtcgagtgg tcagtcctcg gtcagcagcg gggcgggcgt caacaggctc gacttcattt
     1981 tcggtattgg ctggcacaga gtccacccca aaagggtgcc acggctggag gtcatccagg
     2041 ctgaattccc cattcacggg aaggagctcc acggtggtct tggtttccgt cagcgaaggc
     2101 atgagagcgt cgtttccgta gctgattctg ggctcactga tcatgttggc caagacatcg
     2161 tcggagtagt tctgctcctt ctgaagcagc tcatcgactt catcttgaat ctcctcagcc
     2221 accgcaggga cattgtagag cagggacaga gactggttca tgcgctcgta gatcacacgg
     2281 aggtgtgtca taacctggga ccggatctga gcagctttct tggggtccac catgcgcaca
     2341 tgttcaaaat gctttagggt gtgctgtctg tctttctgct ccgcacggac gtacttcttc
     2401 agcatgttga acacatgatg aggccttggg ggcaccgcct gcagtgcagt gatgtaattc
     2461 tcgagggcca ggcggcggcg gtcattgagc atggcttcaa ctctggccat gtgtgtctct
     2521 acaagctgct gtctctcatt ggctgcttcc tgttccagag attccacttt ctcctggaaa
     2581 tgctggataa cggccttctt gtcagctttg ggcaagttct tggcttgacg ctctgcctct
     2641 tcccattctc tcatgacctg ggacattctc tctcggtgct tggcttccag cctctctttg
     2701 gctttctgga aatgggcatg ctcgttctcg tccccgggtg tctccaggta cttgtcgacg
     2761 gcgtcggggg tgctggctgc tgtcgtggga aacacgctgc cacacaccgc catgcagtac
     2821 tcttccgtgt caaagttgtt cctgttgccg ccacatccgc cgtaaaagaa tgggacacac
     2881 ttcccttcag tgacatcaaa gtaccagcgg gagatcattg cgcggcatgg cccggtctcg
     2941 gcttgttcag agcacacctc tcggaccacc tcctccacgg actcagtggt ggttgtggtg
     3001 gtggtggcag tgctggttgt tctctcggtg gcctcttcgt agggctcctc ggcctcctcc
     3061 tccacctcgt ccccatcctc cacatcctca tcatcatcag cttcctcttc ctcaacatca
     3121 gccacttcct cctcttcggc gacttctact actttgtctt caccgccatc agcgtagtct
     3181 gtgtccgctc caccccacca gacatcagag tcatcctcct ctgcatccgc agaatccacg
     3241 ctgtcgcttt cctcggccaa cgggcagcat acaaactcta cccctcggaa cttgtcgatg
     3301 ccgcagggca gcagcatgcc atagtcgtgc aagttagtgc tcttctcgct gcatgtctct
     3361 ttggcgacgg tgtgccagtg aagatgggtc tcacaaacat ccatccgctc ctggtgtagg
     3421 aacttgcact tgtcgggcac gagaagggcg tcgctcacaa actcaccaac taggcaacgg
     3481 taaggaatca cgatgtgggt gtgtgtcttg cactgcttgc ggccccgctt gcaccagttc
     3541 tggatggtca ctggctggtt ggcttccacc acgtttgtga tctgcagttc agggtagacc
     3601 tcttggcagt actgcaagat gccctccttg gtgccaatgc aggttttggt ccctgacggg
     3661 tctgactccc actttccatt ctgcacattc atgtgcatgt tgagtttacc acagaacatg
     3721 gcgatctggg gttctgccag cagcccggcg ttgccatcag tgggtacctc cagagcccga
     3781 accgtccagg cggccagcag gagcagtgcc aagctgggca gcatgctagc ataacttcgt
     3841 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     3901 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc
     3961 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4021 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4081 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     4141 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     4201 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4261 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4321 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     4381 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     4441 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     4501 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4561 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4621 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4681 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4741 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     4801 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     4861 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     4921 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     4981 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     5041 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     5101 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     5161 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     5221 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5281 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5341 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5401 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5461 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5521 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5581 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5641 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5701 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5761 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5821 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5881 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5941 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     6001 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     6061 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6121 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6181 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6241 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6301 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6361 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6421 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6481 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6541 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6601 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6661 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6721 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6781 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6841 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6901 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6961 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     7021 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     7081 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     7141 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7201 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7261 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7321 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7381 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7441 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7501 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7561 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7621 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7681 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7741 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7801 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
