LOCUS       sequence-483648-        7165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Amplifier AAV vector of Dual-AAV sparse labeling system encoding 
            membrane-tethered seTurboID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7165)
  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 7165)
  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-mem-seTurboID-IRES-tTA
FEATURES             Location/Qualifiers
     source          1..7165
                     /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"
     protein_bind    complement(3185..3232)
                     /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(3234..3267)
                     /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    3305..3893
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3358..3378)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3776..3787)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3912..4388
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4111..4130)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4428..4568
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4428..4557
                     /label=AAV2 ITR
     rep_origin      4643..5098
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4730..4749)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4940..4961
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5115..5134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5234..5256
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5294..5312)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5380..5484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5485..5553,5554..6345)
                     /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      6516..7104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7005..7024
                     /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 tcgatctttt
     3121 ggtcctcatc attcttttca acctgtttgg ttcttctcat acagcacagc atggtggcgc
     3181 tagcgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcggaagttc
     3241 ctatactatt tgaagaatag gaacttcgga ataggaactt caattcgata tcaagcttat
     3301 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3361 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3421 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3481 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3541 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3601 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3661 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     3721 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     3781 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3841 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     3901 ccgagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     3961 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     4021 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     4081 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     4141 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     4201 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     4261 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     4321 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     4381 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     4441 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4501 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4561 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4621 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4681 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     4741 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4801 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4861 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4921 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     4981 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5041 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5101 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5161 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5221 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5281 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5341 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5401 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5461 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5521 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5581 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5641 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5701 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5761 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5821 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5881 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5941 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6001 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6061 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6121 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6181 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6241 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6301 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6361 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6421 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6661 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6901 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7081 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7141 ttttgctggc cttttgctca catgt
//
