LOCUS       sequence-481793-        7108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Amplifier AAV vector of Dual-AAV sparse labeling system encoding 
            seTurboID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7108)
  AUTHORS   Zhong S, Zhang X, Gao X, Li Z, Huang L, Guo Q, Gong R, Ren J, Luo M,
            Lin R
  TITLE     Ultrabright chemical labeling enables rapid neural connectivity 
            profiling in large tissue samples.
  JOURNAL   Neuron. 2025 Sep 18:S0896-6273(25)00657-9. doi: 
            10.1016/j.neuron.2025.08.022.
  PUBMED    40972576
REFERENCE   2  (bases 1 to 7108)
  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: "Neuron. 
            2025 Sep 18:S0896-6273(25)00657-9. doi: 
            10.1016/j.neuron.2025.08.022."
COMMENT     Sequence Label: pAAV-TRE-fDIO-seTurboID-IRES-tTA
FEATURES             Location/Qualifiers
     source          1..7108
                     /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(1940..2101)
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="YKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
                     ATKTFTVTE"
     CDS             complement(2117..3073)
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             complement(3089..3112)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3112..3121
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3128..3175)
                     /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(3177..3210)
                     /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    3248..3836
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3301..3321)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3719..3730)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3855..4331
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4054..4073)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4371..4511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4371..4500
                     /label=AAV2 ITR
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5177..5199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5237..5255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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"
     rep_origin      6459..7047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6948..6967
                     /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 aggggcttat tcggttaccg tgaaggtttt ggtagcgtcg tcgtaggtcc
     1981 attcaccgtc aacaccgttg tcgttagcgt actgtttgaa aactttttcc gcggtagcag
     2041 cgtcaacagc ttcggtggtg gtttcacctt tcagggtttt accgttcagg ataagcttgt
     2101 aagatccgcc accgcccttt tcggcagacc gcagactgat ttctccgccc atccagggtt
     2161 tgataactcc gtcctgttcc agcagcaggg ctccctgttt atcaatcccc cggctaatcc
     2221 cgaaaatctc tttgtcccca atgatcagct tcacgggtct attgatgaag ttatccagct
     2281 tctcccaccg tggcagatat ggagccaggc cttcctgctc gaacagttcc agggctgccc
     2341 gcagctctcg gatcagcgta gcggccagag tattcctgtc caggttaatc cctgcttcct
     2401 gcagtgtgat ccagccctga ttgaccacgc tttcctccac gcgcctcata gccacgttaa
     2461 tcccggcgcc aatgacgatc tgtgcagcat cgcctgttat tccggccagc tccaccagga
     2521 tgcctgccag ctttctatcc tgcagataca ggtcattggg ccatttgact cgcaccttgt
     2581 ctgctcccag ctttcgcagc gcttctgcca tgacaattcc gatgaccggg cccaggccga
     2641 ttgctgctgg tccccgcttc aggcgccaaa acatactcag gtacaggtta gctccaaaag
     2701 gagagaacca tttcctccct ctgcttcctc tccctgcctg ctgatattct gcaatgcaag
     2761 catccccact cttcagctcg ccgattcgat ccagcaggta ctgattggtg gagtcgacca
     2821 caggcaggac tgccacgctc ccgccgtcca gctgtcccag aatctgttta gcgttcagca
     2881 gcgggatagg ctcgggcaga gagtagccct ttccgggcac ggtgaacacg tccacgcccc
     2941 agtcgcgcag agtctgaatg tgcttgttga tagcggccct ggacatgccc agggtttctc
     3001 ccagctgttc gccactatgg aactcgccat tagccaggag agcgatcagc ttcagaggca
     3061 cagtattgtc tttgctagcc atggatccct tatcgtcgtc atccttgtaa tccatggtgg
     3121 cgctagcgaa gttcctatac tttctagaga ataggaactt cggaatagga acttcggaag
     3181 ttcctatact atttgaagaa taggaacttc ggaataggaa cttcaattcg atatcaagct
     3241 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3301 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3361 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3421 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3481 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3541 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3601 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3661 gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3721 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3781 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     3841 ataccgagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     3901 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     3961 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     4021 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     4081 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     4141 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     4201 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     4261 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     4321 tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct
     4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4501 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7081 gccttttgct ggccttttgc tcacatgt
//
