LOCUS       sequence-394296-        5460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of tTA under the CAG promoter in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5460)
  AUTHORS   Leiwe MN, Fujimoto S, Baba T, Moriyasu D, Saha B, Sakaguchi R, 
            Inagaki S, Imai T
  TITLE     Automated neuronal reconstruction with super-multicolour Tetbow 
            labelling and threshold-based clustering of colour hues.
  JOURNAL   Nat Commun. 2024 Jun 25;15(1):5279. doi: 10.1038/s41467-024-49455-y.
  PUBMED    38918382
REFERENCE   2  (bases 1 to 5460)
  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: "Nat 
            Commun."; date: "2024-06-25"; volume: "15(1):5279. doi"; pages: " 
            10.1038/s41467-024-49455-y"
COMMENT     Sequence Label: pAAV-CAG-tTA
FEATURES             Location/Qualifiers
     source          1..5460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR
     promoter        442..719
                     /label=chicken beta-actin promoter
     primer_bind     998..1020
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1082..1101
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1146..1763,1764..1769,1770..1805,1806..1808,1809..1844,
                     1845..1847,1848..1883,1884..1892)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    1905..2493
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1958..1978)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2376..2387)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2536..2657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2547..2566)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2601..2620
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   2819..2948
                     /label=AAV2 ITR
     rep_origin      complement(3185..3773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3265..3284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3944..4735,4736..4804))
                     /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     4567..4586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4805..4909)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4935..5390)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5072..5093)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5284..5303
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt agccatgctc
      181 taggaagagt accattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      241 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      301 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      361 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      421 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      481 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      541 tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
      601 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      661 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      721 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      781 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      841 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      901 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
      961 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1021 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1081 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccggtaccg
     1141 ccaccatgtc tagactggac aagagcaaag tcataaactc tgctctggaa ttactcaatg
     1201 aagtcggtat cgaaggcctg acgacaagga aactcgctca aaagctggga gttgagcagc
     1261 ctaccctgta ctggcacgtg aagaacaagc gggccctgct cgatgccctg gcaatcgaga
     1321 tgctggacag gcatcatacc cacttctgcc ccctggaagg cgagtcatgg caagactttc
     1381 tgcggaacaa cgccaagtca ttccgctgtg ctctcctctc acatcgcgac ggggctaaag
     1441 tgcatctcgg cacccgccca acagagaaac agtacgaaac cctggaaaat cagctcgcgt
     1501 tcctgtgtca gcaaggcttc tccctggaga acgcactgta cgctctgtcc gccgtgggcc
     1561 actttacact gggctgcgta ttggaggatc aggagcatca agtagcaaaa gaggaaagag
     1621 agacacctac caccgattct atgcccccac ttctgagaca agcaattgag ctgttcgacc
     1681 atcagggagc cgaacctgcc ttccttttcg gcctggaact aatcatatgt ggcctggaga
     1741 aacagctaaa gtgcgaaagc ggcgggccgg ccgacgccct tgacgatttt gacttagaca
     1801 tgctcccagc cgatgccctt gacgactttg accttgatat gctgcctgct gacgctcttg
     1861 acgattttga ccttgacatg ctccccgggt aaaagcttat cgataatcaa cctctggatt
     1921 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     1981 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2041 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2101 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2161 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     2221 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     2281 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     2341 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     2401 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     2461 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc gggcggccgc
     2521 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     2581 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     2641 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
     2701 agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg
     2761 atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag
     2821 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     2881 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     2941 gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3001 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3061 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3361 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3601 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4921 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     4981 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     5041 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     5101 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     5161 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     5221 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     5281 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     5341 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     5401 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
//
