LOCUS       sequence-479124-        7327 bp    DNA     circular SYN 21-JUL-2025
DEFINITION  The plasmid will package wild-type human Tau (hMAPT) as a T2A fusion
            with EGFP from a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7327)
  AUTHORS   Steele Lab
  TITLE     pAAV CMV-hTau-T2A-EGFP-WPRE3-BGHpA
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7327)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV CMV-hTau-T2A-EGFP-WPRE3-BGHpA
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        218..521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..725
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     676..696
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    746..770
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      771..780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3285..3338
                     /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"
     CDS             join(3339..3341,3342..4055)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3339..3341,3342..4052)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3381..3402)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3642..3661)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3989..4010
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4056..4080)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4141..4161)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(4358..4375)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4364..4571
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4579..4719
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4579..4708
                     /label=AAV2 ITR
     rep_origin      4794..5249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4881..4900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5091..5112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5266..5285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5385..5407
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5445..5463)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5531..5635
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5636..5704,5705..6496)
                     /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(5854..5873)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6667..7255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7156..7175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7317..7327,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgtag ttattaatag taatcaatta
      181 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      241 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      301 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      361 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      421 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      481 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      541 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      601 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      661 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      721 gagctggttt agtgaaccgt cagatccaag tttgtacaaa aaagcaggct gccaccatgg
      781 ctgagccccg ccaggagttc gaagtgatgg aagatcacgc tgggacgtac gggttggggg
      841 acaggaaaga tcaggggggc tacaccatgc accaagacca agagggtgac acggacgctg
      901 gcctgaaaga atctcccctg cagaccccca ctgaggacgg atctgaggaa ccgggctctg
      961 aaacctctga tgctaagagc actccaacag cggaagctga agaagcaggc attggagaca
     1021 cccccagcct ggaagacgaa gctgctggtc acgtgaccca agaggagttg agagttccgg
     1081 gccggcagag gaaggcgcct gaaaggcccc tggccaatga gattagcgcc cacgtccagc
     1141 ctggaccctg cggagaggcc tctggggtct ctgggccgtg cctcggggag aaagagccag
     1201 aagctcccgt cccgctgacc gcgagccttc ctcagcaccg tcccgtttgc ccagcgcctc
     1261 ctccaacagg aggccctcag gagccctccc tggagtgggg acaaaaaggc ggggactggg
     1321 ccgagaaggg tccggccttt ccgaagcccg ccaccactgc gtatctccac acagagcctg
     1381 aaagtggtaa ggtggtccag gaaggcttcc tccgagagcc aggcccccca ggtctgagcc
     1441 accagctcat gtccggcatg cctggggctc ccctcctgcc tgagggcccc agagaggcca
     1501 cacgccaacc ttcggggaca ggacctgagg acacagaggg cggccgccac gcccctgagc
     1561 tgctcaagca ccagcttcta ggagacctgc accaggaggg gccgccgctg aagggggcag
     1621 ggggcaaaga gaggccgggg agcaaggagg aggtggatga agaccgcgac gtcgatgagt
     1681 cctcccccca agactcccct ccctccaagg cctccccagc ccaagatggg cggcctcccc
     1741 agacagccgc cagagaagcc accagcatcc caggcttccc agcggagggt gccatccccc
     1801 tccctgtgga tttcctctcc aaagtttcca cagagatccc agcctcagag cccgacgggc
     1861 ccagtgtagg gcgggccaaa gggcaggatg cccccctgga gttcacgttt cacgtggaaa
     1921 tcacacccaa cgtgcagaag gagcaggcgc actcggagga gcatttggga agggctgcat
     1981 ttccaggggc ccctggagag gggccagagg cccggggccc ctctttggga gaggacacaa
     2041 aagaggctga ccttccagag ccctctgaaa agcagcctgc tgctgctccg cgggggaagc
     2101 ccgtcagccg ggtccctcaa ctcaaagctc gcatggtcag taaaagcaaa gacgggactg
     2161 gaagcgatga caaaaaagcc aagacatcca cacgttcctc tgctaaaacc ttgaaaaata
     2221 ggccttgcct tagccccaaa caccccactc ctggtagctc agaccctctg atccaaccct
     2281 ccagccctgc tgtgtgccca gagccacctt cctctcctaa atacgtctct tctgtcactt
     2341 cccgaactgg cagttctgga gcaaaggaga tgaaactcaa gggggctgat ggtaaaacga
     2401 agatcgccac accgcgggga gcagcccctc caggccagaa gggccaggcc aacgccacca
     2461 ggattccagc aaaaaccccg cccgctccaa agacaccacc cagctctggt gaacctccaa
     2521 aatcagggga tcgcagcggc tacagcagcc ccggctcccc aggcactccc ggcagccgct
     2581 cccgcacccc gtcccttcca accccaccca cccgggagcc caagaaggtg gcagtggtcc
     2641 gtactccacc caagtcgccg tcttccgcca agagccgcct gcagacagcc cccgtgccca
     2701 tgccagacct gaagaatgtc aagtccaaga tcggctccac tgagaacctg aagcaccagc
     2761 cgggaggcgg gaaggtgcag ataattaata agaagctgga tcttagcaac gtccagtcca
     2821 agtgtggctc aaaggataat atcaaacacg tcccgggagg cggcagtgtg caaatagtct
     2881 acaaaccagt tgacctgagc aaggtgacct ccaagtgtgg ctcattaggc aacatccatc
     2941 ataaaccagg aggtggccag gtggaagtaa aatctgagaa gcttgacttc aaggacagag
     3001 tccagtcgaa gattgggtcc ctggacaata tcacccacgt ccctggcgga ggaaataaaa
     3061 agattgaaac ccacaagctg accttccgcg agaacgccaa agccaagaca gaccacgggg
     3121 cggagatcgt gtacaagtcg ccagtggtgt ctggggacac gtctccacgg catctcagca
     3181 atgtctcctc caccggcagc atcgacatgg tagactcgcc ccagctcgcc acgctagctg
     3241 acgaggtgtc tgcctccctg gccaagcagg gtttgggaag cggagagggc aggggaagtc
     3301 ttctaacatg cggggacgtg gaggaaaatc ccggccccgt gagcaagggc gaggagctgt
     3361 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     3421 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     3481 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     3541 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     3601 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3661 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3721 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     3781 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     3841 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     3901 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     3961 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     4021 ggatcactct cggcatggac gagctgtaca agtgaaccca gctttcttgt acaaagtggg
     4081 aattcataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4141 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4201 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttagttc ttgccacggc
     4261 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4321 caattccgtg gtggaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     4381 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4441 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4501 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     4561 catgctgggg agggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     4621 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     4681 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     4741 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     4801 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4861 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4921 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4981 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     5041 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5101 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     5161 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5221 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     5281 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5341 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5401 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5461 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5521 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5581 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5641 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5701 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5761 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5821 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5881 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5941 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6001 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6061 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6121 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6181 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6241 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6301 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     6361 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6421 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6481 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6541 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6601 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6661 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6721 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6781 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6841 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6901 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6961 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7021 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7081 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7141 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7201 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7261 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg
     7321 caggcag
//
