LOCUS       sequence-311049-        5787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated and Cre-dependent expression of tTA under the CAG 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5787)
  AUTHORS   Leiwe MN, Fujimoto S, Baba T, Moriyasu D, Saha B, Sakaguchi R, 
            Inagaki S, Imai T
  TITLE     Automated neuronal reconstruction with super-multicolour 
            fluorescence imaging
  JOURNAL   bioRxiv 2022.10.20.512984
REFERENCE   2  (bases 1 to 5787)
  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: "bioRxiv 
            2022.10.20.512984"
COMMENT     Sequence Label: pAAV-FLEX-tTA
FEATURES             Location/Qualifiers
     source          1..5787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     promoter        441..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1135..1168
                     /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    1231..1264
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1289..1297,1298..1333,1334..1336,1337..1372,
                     1373..1375,1376..1411,1412..1417,1418..2035))
                     /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"
     protein_bind    complement(2052..2085)
                     /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(2156..2189)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2231..2819
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2284..2304)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2702..2713)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2862..2983
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2873..2892)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2927..2946
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3145..3274
                     /label=AAV2 ITR
     rep_origin      complement(3511..4099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3591..3610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4270..5061,5062..5130))
                     /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     4893..4912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5131..5235)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5261..5716)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5398..5419)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5610..5629
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
      181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
      481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
      541 gggggcgggg gggggggggg ggcgcgcgcc gggggggggg gggggggggg gggggggggg
      601 gggggcgggg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccccataact
     1141 tcgtataaag tatcctatac gaagttatat caaaatagga agaccaatgc ttcaccatcg
     1201 acccgaattg ccaagcatca ccatcgaccc ataacttcgt ataatgtatg ctatacgaag
     1261 ttatactagg gatcccctta cttgtacatt acccggggag catgtcaagg tcaaaatcgt
     1321 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     1381 tgtctaagtc aaaatcgtca agggcgtcgg ccggcccgcc gctttcgcac tttagctgtt
     1441 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgat
     1501 ggtcgaacag ctcaattgct tgtctcagaa gtgggggcat agaatcggtg gtaggtgtct
     1561 ctctttcctc ttttgctact tgatgctcct gatcctccaa tacgcagccc agtgtaaagt
     1621 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     1681 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     1741 ctttagcccc gtcgcgatgt gagaggagag cacagcggaa tgacttggcg ttgttccgca
     1801 gaaagtcttg ccatgactcg ccttccaggg ggcagaagtg ggtatgatgc ctgtccagca
     1861 tctcgattgc cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     1921 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactt
     1981 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatggtac
     2041 cgaattctag tataacttcg tataggatac tttatacgaa gttatcattg ggattcttcc
     2101 tattttgatc caagcatcac catcgaccct ctagtccaga tctcaccatc gacccataac
     2161 ttcgtatagc atacattata cgaagttatg tccctcgaag aggttcgaat tcgatatcaa
     2221 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2281 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2341 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     2401 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     2461 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     2521 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     2581 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     2641 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     2701 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     2761 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     2821 tcgataccgt cgacccgggc ggccgcttcg agcagacatg ataagataca ttgatgagtt
     2881 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     2941 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3001 tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct
     3061 ctacaaatgt ggtaaaatcg ataaggatct tcctagagca tggctacgta gataagtagc
     3121 atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc
     3181 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     3241 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat taatgaatcg gccaacgcgc
     3301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3361 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3421 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3481 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3541 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3601 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3661 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3721 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3781 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3841 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3901 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3961 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4021 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4081 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4141 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4201 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4261 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4321 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4381 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4441 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4501 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4561 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4621 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4681 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4741 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4801 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4861 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4921 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4981 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5041 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5101 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5221 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5281 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5341 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     5401 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     5461 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     5521 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     5581 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     5641 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     5701 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     5761 gtgcgggcct cttcgctatt acgccag
//
