LOCUS       sequence-234735-        5930 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for sparse and bright labeling in a cell-type specific 
            manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5930)
  AUTHORS   Lin R, Wang R, Yuan J, Feng Q, Zhou Y, Zeng S, Ren M, Jiang S, Ni H,
            Zhou C, Gong H, Luo M
  TITLE     Cell-type-specific and projection-specific brain-wide reconstruction
            of single neurons.
  JOURNAL   Nat Methods. 2018 Nov 19. pii: 10.1038/s41592-018-0184-y. doi: 
            10.1038/s41592-018-0184-y.
  PUBMED    30455464
REFERENCE   2  (bases 1 to 5930)
  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 
            Methods. 2018 Nov 19. pii: 10.1038/s41592-018-0184-y. doi: 
            10.1038/s41592-018-0184-y."
COMMENT     Sequence Label: pAAV-TRE-DIO-FLPo
FEATURES             Location/Qualifiers
     source          1..5930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        148..462
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    156..174
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    263..281
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    299..317
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    334..352
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    370..388
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     435..459
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(491..524)
                     /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(575..608)
                     /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(617..1888,1889..1909,1910..1915))
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     regulatory      1912..1921
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1928..1961
                     /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    2012..2045
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2070..2658
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2123..2143)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2541..2552)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2677..3153
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2876..2895)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3193..3333
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3193..3322
                     /label=AAV2 ITR
     rep_origin      3408..3863
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3495..3514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3705..3726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3880..3899)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3999..4021
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4059..4077)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4145..4249
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4250..4318,4319..5110)
                     /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(4468..4487)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5281..5869
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5770..5789
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctcgag tttactccct atcagtgata gagaacgtat
      181 gtcgagttta ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat
      241 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
      301 cctatcagtg atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt
      361 cgagtttact ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag
      421 gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagaat tctctagagt
      481 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
      541 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
      601 gaagttatgg cgcgcctcag atccgcctgt tgatgtagct gctcaggtag tccagcacct
      661 cctggctgat gatgccgttc caggcggggt atctgatgct gccctcggcg ctgcccttca
      721 gctgctcgat gtgctgccac tcctcgatgg ggttggtctc gtccttcagg gcgatcatct
      781 ccttgctgat ggggtcgtag gcgtagtacc tggacaccag ggcgaagtag tggtcgggga
      841 tggcggtgat ctggtgggtg taggtggtcc tggccacggc ggaggccctc ttgtcgctcc
      901 agttgcccac cacgtttgtc agctcggtca ggcccttcat gctcagaaag ctggtcatca
      961 ggtgcctgcc gatgtggctc ttagggccgt tcttgatagc gaagatgggg tagggggcgt
     1021 tcttcttcag ggccttgttg tagctgcgca ccaggttgtc cttcagcagc tggtactcct
     1081 gcttgttgct gctgctgttg ccggtcctgt tcactctctt cagcacgggc tcgctgttcc
     1141 tcaggaactc gtccaggtac accagggggt cgatcctgcc tctggcgctg aaaaagtaga
     1201 tgtgcctgga cacgcttgtc ttggtctcgg tcaccaggca ctgaatgatc acgcccaggt
     1261 acttgttctg caccagcttg aagctcttgg ggtccacgtt cttgatgtcg ctgaacctgc
     1321 cgcagttgat gaatgtggcc aggaacagga actggtacag ggtcttggtc ttggtgaacc
     1381 tgctggtgta ctcgaagctg ttcaggatct tctcggtgat ctcccagatg ctctcgccct
     1441 cggacagcag ggccttcagc atcttcttgc tgtggctgtt gcccttgtcg gcctcctcgc
     1501 tgctctcgaa ctgcagctgc aggctggaca cgatgtcggt gatgtcgctc tggtgcttct
     1561 ggccgttgta agggatgatg gtgaactccc aggcggggat cagcttcttc aggctggcct
     1621 ccaggatggt ggccttctgg gtcttgtact tgaactgcag gctcttgttc acgatgtcga
     1681 agctcaggct gttgctgatg atggtgttgt agctcatgaa ggtggccctc ttgatggcgg
     1741 tgccgttgtg ggtgatcatc cagcacaggt aggtcagctc ggcggcacag ctggcgatct
     1801 tctcgccgct gggcctctcg aatctctcca cgaactgccg caccagcacc ttgggggggg
     1861 tcttgcacag gatgtcgaac tggctcatca ccttcctctt cttcttagga gccatggtgg
     1921 cgctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt
     1981 atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa
     2041 gttatgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     2101 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     2161 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     2221 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     2281 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     2341 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     2401 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     2461 ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga
     2521 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     2581 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     2641 tttgggccgc ctccccgcat cgataccgag atctacgggt ggcatccctg tgacccctcc
     2701 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     2761 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     2821 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     2881 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     2941 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     3001 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     3061 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     3121 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga
     3181 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     3241 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     3301 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     3361 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     3421 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3481 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3541 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3601 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     3661 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3721 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     3781 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     3841 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     3901 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3961 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4021 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     4081 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4141 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4201 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     4261 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4321 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4381 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4441 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     4501 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4561 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4621 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4681 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4741 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     4801 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4861 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4921 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4981 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5041 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5101 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5161 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5221 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5281 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5341 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5401 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5461 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5521 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5581 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5641 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5701 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5761 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5821 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5881 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
