LOCUS       sequence-227787-        6649 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 6649)
  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 6649)
  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-fDIO-GFP-IRES-tTA
FEATURES             Location/Qualifiers
     source          1..6649
                     /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    493..540
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    563..596
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(join(605..613,614..649,650..652,653..688,
                     689..691,692..727,728..733,734..1351))
                     /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    1360..1912
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1362..1935
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1522..1541)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1751..1768
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(join(1937..2650,2651..2653,2654..2656))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1982..2003)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2331..2350
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2590..2611
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2653..2662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2669..2716)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(2718..2751)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    2789..3377
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2842..2862)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3260..3271)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3396..3872
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3595..3614)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3912..4052
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3912..4041
                     /label=AAV2 ITR
     rep_origin      4127..4582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4214..4233)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4424..4445
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4599..4618)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4718..4740
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4778..4796)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4864..4968
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4969..5037,5038..5829)
                     /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(5187..5206)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6000..6588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6489..6508
                     /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 tctgtcgtga
      481 ggtaccggat ccgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
      541 ttaattaaga agttcctatt ccgaagttcc tattcttcaa atagtatagg aacttcggcg
      601 cgccttaccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat
      661 caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg
      721 cgtcggccgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt
      781 ccaggccgaa aaggaaggca ggttcggctc cctgatggtc gaacagctca attgcttgtc
      841 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat
      901 gctcctgatc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca
      961 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt
     1021 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga
     1081 ggagagcaca gcggaatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt
     1141 ccagggggca gaagtgggta tgatgcctgt ccagcatctc gattgccagg gcatcgagca
     1201 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag
     1261 cgagtttcct tgtcgtcagg ccttcgatac cgacttcatt gagtaattcc agagcagagt
     1321 ttatgacttt gctcttgtcc agtctagaca ttgtggccat attatcatcg tgtttttcaa
     1381 aggaaaacca cgtccccgtg gttcgggggg cctagacgtt tttttaacct cgactaaaca
     1441 catgtaaagc atgtgtaccg aggccccaga tcagatccca tacaatgggg taccttctgg
     1501 gcatccttca gccccttgtt gaatacgctt gaggagagcc atttgactct ttccacaact
     1561 atccaactca caacgtggca ctggggttgt gccgcctttg caggtgtatc ttatacacgt
     1621 ggcttttggc cgcagaggca cctgtcgcca ggtggggggt tccgctgcct gcaaagggtc
     1681 gctacagacg ttgtttgtct tcaagaagct tccagaggaa ctgcttcctt cacgacattc
     1741 aacagacctt gcattccttt ggcgagaggg gaaagacccc taggaatgct cgtcaagaag
     1801 acagggccag gtttccgggc cctcacattg ccaaaagacg gcaatatggt ggaaaataac
     1861 atatagacaa acgcacaccg gccttattcc aagcggcttc ggccagtaac gttagggggg
     1921 ggggagggag aggggcttac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1981 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
     2041 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     2101 tctgctggta gtggtcggcg agctgcacgc cgccgtcctc gatgttgtgg cggatcttga
     2161 agttcgcctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
     2221 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     2281 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     2341 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     2401 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     2461 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
     2521 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     2581 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     2641 cgcccttgct caccatggtg gcgctagcga agttcctata ctttctagag aataggaact
     2701 tcggaatagg aacttcggaa gttcctatac tatttgaaga ataggaactt cggaatagga
     2761 acttcaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     2821 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2881 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2941 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3001 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3061 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3121 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3181 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
     3241 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3301 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3361 ttgggccgcc tccccgcatc gataccgaga tctacgggtg gcatccctgt gacccctccc
     3421 cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa
     3481 aattaagttg catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg
     3541 gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt
     3601 gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg
     3661 ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca
     3721 ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg
     3781 tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac
     3841 aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac
     3901 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3961 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4021 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4081 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4141 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4201 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4261 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4321 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4381 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4441 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4501 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4561 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4621 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4681 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4741 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4801 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     4861 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4921 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     4981 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5041 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5101 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5161 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5221 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5281 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5341 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5401 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5461 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5521 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5581 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5641 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5701 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5761 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     5821 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     5881 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5941 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6001 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6061 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6121 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6541 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6601 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
