LOCUS       sequence-470391-        6906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcriptional reporter for detecting calcium transients in 
            neuronal cultures (transcription factor) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6906)
  AUTHORS   Zhou G, Li R, Bartolik O, Ma Y, Wan WW, Meng J, Hu Y, Ye B, Wang W
  TITLE     An improved FLARE system for recording and manipulating neuronal 
            activity.
  JOURNAL   Cell Rep Methods. 2025 Apr 21;5(4):101012. doi: 
            10.1016/j.crmeth.2025.101012. Epub 2025 Mar 21.
  PUBMED    40120579
REFERENCE   2  (bases 1 to 6906)
  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: "Cell Rep 
            Methods."; date: "2025-04-21"; volume: "5(4):101012. doi"; pages: " 
            10.1016/j.crmeth.2025.101012. Epub 2025 Mar 21"
COMMENT     Sequence Label: pAAV-cytoFLARE1.0/1.1-TF
FEATURES             Location/Qualifiers
     source          1..6906
                     /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        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             657..1592
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             2205..2225
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2238..2261
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2262..2882,2883..3269)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2335..2354)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     misc_feature    3297..3885
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3350..3370)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3768..3779)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3927..3944)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3933..4140
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4169..4309
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4169..4298
                     /label=AAV2 ITR
     rep_origin      4384..4839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4471..4490)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4681..4702
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4856..4875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4975..4997
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5035..5053)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5121..5225
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5226..5294,5295..6086)
                     /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(5444..5463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6257..6845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6746..6765
                     /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 gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg ccatctgctg
      661 gagacatgag agctgccaac ctttggccaa gcccgctcat gatcaaacgc tctaagaaga
      721 acagcctggc cttgtccctg acggccgacc agatggtcag tgccttgttg gatgctgagc
      781 cccccatact ctattccgag tatgatccta ccagaccctt cagtgaagct tcgatgatgg
      841 gcttactgac caacctggca gacagggagc tggttcacat gatcaactgg gcgaagaggg
      901 tgccaggctt tgtggatttg accctccatg atcaggtcca ccttctagaa tgtgcctggc
      961 tagagatcct gatgattggt ctcgtctggc gctccatgga gcacccagtg aagctactgt
     1021 ttgctcctaa cttgctcttg gacaggaacc agggaaaatg tgtagagggc atggtggaga
     1081 tcttcgacat gctgctggct acatcatctc ggttccgcat gatgaatctg cagggagagg
     1141 agtttgtgtg cctcaaatct attattttgc ttaattctgg agtgtacaca tttctgtcca
     1201 gcaccctgaa gtctctggaa gagaaggacc atatccaccg agtcctggac aagatcacag
     1261 acactttgat ccacctgatg gccaaggcag gcctgaccct gcagcagcag caccagcggc
     1321 tggcccagct cctcctcatc ctctcccaca tcaggcacat gagtaacaaa ggcatggagc
     1381 atctgtacag catgaagtgc aagaacgtgg tgcccctcta tgacctgctg ctggaggcgg
     1441 cggacgccca ccgcctacat gcgcccacta gccgtggagg ggcatccgtg gaggagacgg
     1501 accaaagcca cttggccact gcgggctcta cttcatcgca ttccttgcaa aagtattaca
     1561 tcacggggga ggcagagggt ttccctgcca cagctgctag cttcaacgct cgcaggaagc
     1621 tgaagggtgc tatcctgttc accatgctcg ccacaagaaa cttcagcggc agctttaatg
     1681 cgcggcgcaa gctaaaggga gccatacttt tcacaatgtt ggctaccaga aatttttcgg
     1741 agctggccga gaagctggcc ggcctggaca tcaacggtgg aactagtgag tttcgggcaa
     1801 ccacactgga acggatcgag aaaagcttcg tgattactga tccgagactg cctgacaacc
     1861 cagtgatttt tgtgagcgat tccttcctgc agctgacaga atattctcgg gaagagatcc
     1921 tggggcgcaa ttgccgtttt ctgcagggac ccgagacaga ccgtgccact gttcggaaaa
     1981 tcagagatgc tattgacaac cagactgaag tgaccgttca gctgatcaat tataccaaga
     2041 gcggcaagaa gttctggaac gtgttccacc tgcagccgat gcgcgattat aagggcgacg
     2101 tccagtactt cattggcgtg cagctggatg gcaccgaacg tcttcatggc gccgctgagc
     2161 gtgaggcggt ctgcctggtc aaaaagacag cctttcagat tgctgagaac ctgtacttcc
     2221 agatgggcgg aggatccgac tacaaggatg acgacgataa gatgtctaga ttagataaaa
     2281 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     2341 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     2401 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     2461 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     2521 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     2581 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     2641 tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg
     2701 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     2761 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     2821 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     2881 ccgcgtacag ccgcgcgcgt acgaaaaaca attacgggtc taccatcgag ggcctgctcg
     2941 atctcccgga cgacgacgcc cccgaagagg cggggctggc ggctccgcgc ctgtcctttc
     3001 tccccgcggg acacacgcgc agactgtcga cggccccccc gaccgatgtc agcctggggg
     3061 acgagctcca cttagacggc gaggacgtgg cgatggcgca tgccgacgcg ctagacgatt
     3121 tcgatctgga catgttgggg gacggggatt ccccgggtcc gggatttacc ccccacgact
     3181 ccgcccccta cggcgctctg gatatggccg acttcgagtt tgagcagatg tttaccgatg
     3241 cccttggaat tgacgagtac ggtgggtaat aggaattcga tatcaagctt atcgataatc
     3301 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3361 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3421 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3481 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3541 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3601 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3661 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3721 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3781 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3841 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3901 tgctcgagct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3961 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4021 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4081 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     4141 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc
     4201 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4261 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4321 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4381 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4441 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4501 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4561 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     4621 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4681 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     4741 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     4801 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     4861 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4921 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4981 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5041 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5101 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5161 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5221 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5281 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5341 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5401 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5461 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5521 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5581 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5641 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5701 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5761 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5821 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5881 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5941 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6001 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6061 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6121 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6181 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6241 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6301 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6361 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6421 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6481 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6541 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6601 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6661 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6721 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6781 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6841 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6901 acatgt
//
