LOCUS       pAAV-hSyn-fDIO-t        6430 bp DNA     circular SYN 11-JUL-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6430)
  AUTHORS   Hagihara
  TITLE     Direct Submission
  JOURNAL   Exported Aug 23, 2023 from SnapGene Viewer 7.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        137..584
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     466..483
                     /label=s20_hSyn-F2
                     /note="color: 黒; sequence: acgcgaggcgcgagatag; added: 
                     2021-10-25"
     protein_bind    622..655
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
                     /note="color: #31849b; direction: RIGHT"
     misc_feature    706..739
                     /label=F5
                     /note="color: #31849b; direction: RIGHT"
     primer_bind     731..772
                     /label=TEV_arm-R
                     /note="tm=61℃"
                     /note="color: 青; sequence: 
                     aggaacttcggcgcgcCTCAAAGCTGAGTGGCTTCCTTAACG; added: 
                     2021-10-03"
     misc_feature    731..745
                     /label=homology arm
                     /note="color: #a6acb3"
     CDS             complement(748..1452)
                     /codon_start=1
                     /label=Tobacco etch virus (TEV) protease
                     /note="color: #ff99cc"
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQL"
     primer_bind     complement(1203..1221)
                     /label=TEVp-286F
                     /note="color: 黒; sequence: GACGGCAGGGACATGATTA; added: 
                     2021-10-25"
     CDS             complement(1453..1506)
                     /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."
                     /note="color: #cc99b2
                     Cleavage site after base 1455"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(1519..2364)
                     /codon_start=1
                     /label=taCasp3
                     /note="color: #00ffff"
                     /translation="MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMG
                     LCIIINNKNFHKSTGMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVS
                     KEDHSKRSSFVCVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQAC
                     RGTELDCGIETENLYFQSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQ
                     SLCAMLKQYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFY
                     H"
     primer_bind     complement(1799..1818)
                     /label=taCasp3-547F
                     /note="color: 黒; sequence: GTTGATGATGACATGGCGTG; added: 
                     2021-10-25"
     CDS             complement(1822..1842)
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /note="color: #cc99b2; direction: LEFT
                     Cleavage site after base 1824"
     primer_bind     complement(2352..2386)
                     /label=taCas3_arm-F
                     /note="Tm=62℃"
                     /note="color: 青; sequence: 
                     taggaacttcgctaGCGCCACCATGGAGAACACTG; added: 2021-10-11"
     regulatory      2361..2370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     misc_feature    2372..2386
                     /label=homology arm
                     /note="color: #a6acb3"
     protein_bind    complement(2377..2410)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
                     /note="color: #31849b; direction: LEFT"
     misc_feature    complement(2461..2494)
                     /label=F5
                     /note="color: #31849b; direction: LEFT"
     misc_feature    2519..3107
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     primer_bind     complement(2572..2592)
                     /label=s47_WPREu-R
                     /note="color: 黒; sequence: catagcgtaaaaggagcaaca; added: 
                     2021-10-25"
     CDS             complement(2990..3001)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 2989"
                     /translation="IEGR"
     polyA_signal    3139..3615
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     repeat_region   3655..3795
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
                     /note="color: #ffe4c4"
     rep_origin      3870..4325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4607..4711
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             4712..5572
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4712 .. 4780 / #ccffcc / signal sequence
                        2: 4781 .. 5572 / #ccffcc
                     Cleavage site after base 4780"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5743..6331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     repeat_region   join(6393..6430,1..92)
                     /label=AAV2 ITR(alternate)
                     /note="color: #ffe4c4"
ORIGIN
        1 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
       61 gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtgtgt ctagactgca
      121 gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg
      181 cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg
      241 cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc
      301 cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc
      361 tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc
      421 accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga
      481 taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt
      541 ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga
      601 tcctctagag tcgactccgg agaagttcct attctctaga aagtatagga acttcgcaga
      661 atggtagctg gattgtagct gctattagca atatgaaacc tcttagaagt tcctattctt
      721 caaaaggtat aggaacttcg gcgcgcctca aagctgagtg gcttccttaa cgggctggaa
      781 gggttcctcg ggcttgacca tgaacacctt atgtccgccc caaagaacgc tgtcggcgtt
      841 cagcctccag ccgctcaccc actgctgcgc ctcttgattg gtcaacagct ccatgaagtt
      901 tttcggcacg ctcgtgaagt agttgttggt attggtaaaa ttgctggcgc tgtggatgcc
      961 cacgatgaag ccgtccctag tgctcaccaa tgggctgccg cattggccat ccttcgtctg
     1021 aatccagtgc ttccagaaga tgccgtcgct gctggggaag gtgcagctgg tatcagagac
     1081 catgctgctc atggacttgg tctggaagtt ggtggtcacc aggcagattc gctcctccct
     1141 ttgtggctcc ctgaacttca gtttctgggg aaaggggggg aagtccttgg gcatcctgat
     1201 gataatcatg tccctgccgt cgatcaggtg ctgttgcagg gtggttgtgt tcttcacttt
     1261 gaacacgccg tgcaggcttt gcaccagcag tgtgccgtta ttcctcctga acagatgctt
     1321 gttggtgatg atgaaggggc cgaagccgat gccgtacaga ctagtggtgt gaccgtcgct
     1381 ctcgttggtc aggtgacaga tggtgctgga gatcgggttg tagtccctcg ggcccttgaa
     1441 aaggctctcg ccagggccgg gattctcctc cacgtcaccg catgttagaa gacttcctct
     1501 gccctctccg cttccggagt gataaaaata gagttctttt gtgagcatgg aaacaataca
     1561 tggaatctgt ttctttgcat gaaaagtagc gtcaaaggaa aaggactcaa attctgttgc
     1621 cacctttcgg ttaacccggg taagaatgtg cataaattca agcttgtcgg catactgttt
     1681 cagcatggca caaagcgact ggatgaacca ggagccatcc tttgaatttc gccaagaata
     1741 ataaccaggt gctgtggagt atgcatacaa gaagtcggcc tccactggta ttttatgaca
     1801 cgccatgtca tcatcaacac cactctggaa gtagagattt tctgtctcaa tgccacagtc
     1861 cagttctgta ccacggcagg cctgaataat gaaaagtttg ggttttccag ttagacttct
     1921 acaacgatcc cctctgaaaa agtttgttat ttttttcagg tcaacaggtc catttgttcc
     1981 aaaaattatt ccttcttcac catggctcag aagcacacaa acaaaactgc tccttttgct
     2041 gtgatcttct ttagaaacat cacgcatcaa ttccacaatt tcttcacgtg taagatcatt
     2101 tttattcctg acttcatatt tcaagtttct gaatgtttcc ctgaggtttg ctgcatcgac
     2161 atctgtacca gaccgagatg tcattccagt gcttttatga aaattcttat tattaattat
     2221 tatacataaa cccatctcag gataatccat tttataactg ttgtccaggg atattccaga
     2281 gtccattgat tcgcttccat gtatgatctt tggttccaaa tttttaatgg attttgaatc
     2341 cactgagttt tcagtgttct ccatggtggc gctagcgaag ttcctatact ttctagagaa
     2401 taggaacttc ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga
     2461 gaagttccta taccttttga agaataggaa cttcgaattc gatatcaagc ttatcgataa
     2521 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     2581 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     2641 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     2701 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     2761 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     2821 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     2881 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     2941 ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     3001 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     3061 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc
     3121 gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     3181 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     3241 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     3301 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     3361 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     3421 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     3481 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     3541 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     3601 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac
     3661 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     3721 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     3781 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3841 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3901 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     3961 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4021 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4081 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4141 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4201 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     4261 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4321 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     4381 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4441 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4501 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     4561 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4621 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4681 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4741 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4801 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4861 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4921 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4981 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5041 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5101 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5161 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5221 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5281 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5341 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5401 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5461 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5521 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5581 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5641 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5701 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5761 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5821 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5881 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5941 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6001 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6061 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6121 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6181 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6241 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6301 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6361 cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct
     6421 cactgaggcc
//
