LOCUS       sequence-410573-        5489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for TRE(TRE3G)-driven smFP_FLAG (FLAG-tagged spaghetti 
            monster) expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5489)
  AUTHORS   Watakabe A, Tani T, Abe H, Skibbe H, Ichinohe N, Yamamori T
  TITLE     Serial Two-Photon Tomography of the Whole Marmoset Brain for 
            Neuroanatomical Analyses.
  JOURNAL   J Vis Exp. 2025 Jan 17;(215). doi: 10.3791/67694.
  PUBMED    39895625
REFERENCE   2  (bases 1 to 5489)
  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: "J Vis 
            Exp."; date: "2025-01-17"; volume: "(215). doi"; pages: " 
            10.3791/67694"
COMMENT     Sequence Label: AAVTRE3_smFP_FLAG
FEATURES             Location/Qualifiers
     source          1..5489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..126
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        143..521
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    151..169
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    187..205
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    223..241
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    259..277
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    295..313
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    331..349
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    367..385
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     430..454
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      544..553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             553..576
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             580..603
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             607..630
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1159..1182
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1189..1212
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1225..1248
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1255..1278
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1489..1512
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1516..1539
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1543..1566
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1616..2204
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1669..1689)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2087..2098)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2236..2712
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2435..2454)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2752..2892
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2752..2881
                     /label=AAV2 ITR
     rep_origin      2967..3422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3054..3073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3264..3285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3439..3458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3558..3580
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3618..3636)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3704..3808
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3809..3877,3878..4669)
                     /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(4027..4046)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4840..5428
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5329..5348
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgcgca gagagggagt ggccaactcc atcactaggg
      121 gttcctgcgg ccgcacgcgt tcgagtttac tccctatcag tgatagagaa cgtatgaaga
      181 gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag
      241 aacgtataag gagtttactc cctatcagtg atagagaacg tatgaccagt ttactcccta
      301 tcagtgatag agaacgtatc tacagtttac tccctatcag tgatagagaa cgtatatcca
      361 gtttactccc tatcagtgat agagaacgta taagctttag gcgtgtacgg tgggcgccta
      421 taaaagcaga gctcgtttag tgaaccgtca gatcgcctgg agcaattcca caacactttt
      481 gtcttatacc aactttccgt accacttcct accctcgtaa agtcgacgaa ttcaattgcg
      541 gccgccacca tggactacaa ggacgacgac gataaaggag attacaaaga tgacgatgat
      601 aagggcgatt ataaggacga cgatgacaag ggcggcgtgt cgaaaggcga ggagctgttc
      661 acaggcgtgg tccccatcct ggttgagctg gacggtgacg ttaacggcca caaatttagc
      721 gtacgtggtg agggcgaggg tgacgctacc aacggaaagc tgaccctgaa attcatctgc
      781 acaactggca agctgccggt gccctggccc acgctggtaa ccactctggg cggaggcgtt
      841 caatgtttca gtcgttaccc ggatcacatg aagcagcatg acttcttcaa gtcggccatg
      901 cccgaaggct acgtgcagga gcgcaccatc agttttaagg atgacggcac ctacaagact
      961 cgcgccgagg tcaagttcga aggagacacc ctggttaatc gtatcgagct caaaggcatc
     1021 gatttcaagg aggacggaaa tattctgggc cacaagctgg agtacaattt taacagccac
     1081 aatgtttaca ttactgccga caagcagaag aacggcatca aggctaattt caagattcgt
     1141 cacaatgtgg agggaggcga ctataaggat gacgatgaca aacaacaaga ctataaggac
     1201 gatgatgaca aaggtcagca aggcgattac aaggacgatg acgataagca gcaagattac
     1261 aaggatgatg atgataaggg aggcgacgga agcgtgcagc tggcagatca ttaccaacag
     1321 aacacgccca ttggtgacgg tccagtcctg ctgcctgata accactactt gtccacccag
     1381 tcggtgctgt cgaaggaccc gaacgagaag cgtgaccaca tggtgctgct ggagttcgtg
     1441 accgccgcag gaatcaccct cggcatggat gagctttaca agggcggaga ttataaagat
     1501 gacgacgaca agggcgacta caaagatgat gatgacaagg gtgactacaa agacgacgat
     1561 gataaataaa ccggtgctcg aggtacggat ccaattcgat atcaagctta tcgataatca
     1621 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     1681 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     1741 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     1801 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     1861 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     1921 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     1981 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta
     2041 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     2101 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     2161 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct
     2221 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg
     2281 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
     2341 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg
     2401 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca
     2461 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct
     2521 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt
     2581 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
     2641 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc
     2701 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc
     2761 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     2821 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     2881 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2941 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     3001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     3061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3181 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3241 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     3301 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     3361 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3421 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3481 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3541 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3601 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3661 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3721 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3781 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3841 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3901 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3961 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4021 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4081 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4141 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4201 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4261 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4321 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4381 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4441 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4501 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4561 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4621 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4681 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4741 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4801 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4861 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4921 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4981 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5041 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5101 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5161 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5221 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5281 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5341 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5401 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5461 ggccttttgc tggccttttg ctcacatgt
//
