LOCUS       sequence-372452-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eGFP-P2A-LgBiT-NLS from human synapsin promoter via 
            adeno-associated virus for neuronal expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  AUTHORS   Tuck BJ, Miller LVC, Katsinelos T, Smith AE, Wilson EL, Keeling S, 
            Cheng S, Vaysburd MJ, Knox C, Tredgett L, Metzakopian E, James LC, 
            McEwan WA
  TITLE     Cholesterol determines the cytosolic entry and seeded aggregation of
            tau.
  JOURNAL   Cell Rep. 2022 May 3;39(5):110776. doi: 
            10.1016/j.celrep.2022.110776.
  PUBMED    35508140
REFERENCE   2  (bases 1 to 5850)
  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.";
            date: "2022-05-3"; volume: "39(5):110776. doi"; pages: " 
            10.1016/j.celrep.2022.110776"
COMMENT     Sequence Label: pAAV-hSyn-eGFP-P2A-LgBiT-NLS
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..135
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        180..627
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      641..650
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(647..649,650..652,653..1363)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(647..649,650..652,653..1363)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(692..713)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(953..972)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1300..1321
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1379..1435
                     /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"
     CDS             1946..1966
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    1982..2570
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2035..2055)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2453..2464)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2602..3078
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2801..2820)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3118..3258
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3118..3247
                     /label=AAV2 ITR
     rep_origin      3333..3788
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3420..3439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3630..3651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3805..3824)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3924..3946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3984..4002)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4070..4174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4175..4243,4244..5035)
                     /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(4393..4412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5206..5794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5695..5714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
       61 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      121 tcactagggg ttcctgcggc cgcacgcgtg tgtctagact gcagagggcc ctgcgtatga
      181 gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac
      241 cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag
      301 agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg
      361 acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg
      421 ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc
      481 gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc
      541 gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga
      601 ggagtcgtgt cgtgcctgag agcgcagtcg agaaggtacc gccaccatgg tgagcaaggg
      661 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      721 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      781 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
      841 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
      901 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
      961 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1021 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1081 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1141 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1201 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1261 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1321 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggaattcg gaagcggagc
     1381 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctggatc
     1441 cgtcttcaca ctcgaagatt tcgttgggga ctgggaacag acagccgcct acaacctgga
     1501 ccaagtcctt gaacagggag gtgtgtccag tttgctgcag aatctcgccg tgtccgtaac
     1561 tccgatccaa aggattgtcc ggagcggtga aaatgccctg aagatcgaca tccatgtcat
     1621 catcccgtat gaaggtctga gcgccgacca aatggcccag atcgaagagg tgtttaaggt
     1681 ggtgtaccct gtggatgatc atcactttaa ggtgatcctg ccctatggca cactggtaat
     1741 cgacggggtt acgccgaaca tgctgaacta tttcggacgg ccgtatgaag gcatcgccgt
     1801 gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga
     1861 cgagcgcctg atcacccccg acggctccat gctgttccga gtaaccatca acagcggagg
     1921 cggaggctca ggaggcggag gctcaccaaa gaagaagcgt aaggtataaa agcttatcga
     1981 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     2041 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     2101 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     2161 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     2221 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     2281 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     2341 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     2401 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     2461 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     2521 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     2581 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     2641 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     2701 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     2761 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     2821 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     2881 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     2941 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     3001 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     3061 gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg
     3121 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     3181 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     3241 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3301 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     3361 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     3421 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3481 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3541 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3601 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3661 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     3721 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     3781 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     3841 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3901 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3961 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     4021 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4081 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4141 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     4201 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4261 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     4321 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     4381 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4441 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     4501 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     4561 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     4621 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     4681 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     4741 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4801 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4861 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4921 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4981 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     5041 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5101 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5161 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5221 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5281 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5341 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5401 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5461 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5521 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5581 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5641 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5701 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5761 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5821 gttcctggcc ttttgctggc cttttgctca
//
