LOCUS       sequence-470400-        6260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcriptional reporter for detecting calcium transients in 
            neuronal cultures (TEVp).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6260)
  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 6260)
  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-TEV
FEATURES             Location/Qualifiers
     source          1..6260
                     /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)"
     regulatory      642..651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(693..714)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(954..973)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1301..1322
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1374..1430
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1875..1916
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    2651..3239
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2704..2724)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3122..3133)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3281..3298)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3287..3494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3523..3663
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3523..3652
                     /label=AAV2 ITR
     rep_origin      3738..4193
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3825..3844)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4035..4056
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4210..4229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4329..4351
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4389..4407)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4475..4579
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4580..4648,4649..5440)
                     /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(4798..4817)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5611..6199
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6100..6119
                     /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 gtgagcaagg
      661 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      721 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      781 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      841 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      901 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      961 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1021 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1081 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1141 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1201 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1261 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1321 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaagc ggagctacta
     1381 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct atggaccaac
     1441 tgactgaaga gcagatcgca gaatttaaag aggctttctc cctactggac aaggacgggg
     1501 atgggacaat aacaaccaag gagctgggga cggggatgcg gtctctgggg cagaacccca
     1561 cagaagcaga gctgcaggac atgatcaatg aagtagatgc cgacggtgac ggcacaatcg
     1621 acttccctga gttcctgaca atgatggcaa gaaaaatgaa atacagggac acggaagaag
     1681 aaattagaga agcgttcggt gtgtttgata aggatggcaa tggctacatc agtgcagcag
     1741 agcttcgcca cgtgatgaca aaccttggag agaagttaac agatgaagag gttgatgaaa
     1801 tgatcaggga agcagacatc gatggggatg gtcaggtaaa ctacgaagag tttgtacaaa
     1861 tgatgacagc gaagggcaag ccgatcccta accctctgct gggcctggac agcactggtg
     1921 gagctagcgg tggaagtgga tcaggcagcg gtggatctgg agaaagcttg tttaagggac
     1981 cacgtgatta caacccgata tcgagcacca tttgtcattt gacgaatgaa tctgatgggc
     2041 acacaacatc gttgtatggt attggatttg gtcccttcat cattacaaac aagcacttgt
     2101 ttagaagaaa taatggaaca ctgttggtcc aatcactaca tggtgtattc aaggtcaaga
     2161 acaccacgac tttgcaacaa cacctcattg atgggaggga catgataatt attcgcatgc
     2221 ctaaggattt cccaccattt cctcaaaagc tgaaatttag agagccacaa agggaagagc
     2281 gcatatgtct tgtgacaacc aacttccaaa ctaagagcat gtctagcatg gtgtcagaca
     2341 ctagttgcac attcccttca tctgatggca tattctggaa gcattggatt caaaccaagg
     2401 atgggcagtg tggcaatcca ttagtatcaa ctagagatgg gttcattgtt ggtatacact
     2461 cagcatcgaa tttcaccaac acaaacaatt atttcacaag cgtgccgaaa aacttcatgg
     2521 aattgttgac aaatcaggag gcgcagcagt gggttagtgg ttggcgatta aatgctgact
     2581 cagtattgtg gggtggccat aaagttttca tggtgtaagg cgcgccgaat tcgatatcaa
     2641 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     2701 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     2761 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     2821 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     2881 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     2941 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3001 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3061 ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3121 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3181 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     3241 tcgataccga gcgctgctcg agctagagct cgctgatcag cctcgactgt gccttctagt
     3301 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3361 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3421 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3481 aggcatgctg gggaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg
     3541 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     3601 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     3661 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     3721 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3781 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     3841 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3901 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3961 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4021 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     4081 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     4141 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     4201 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     4261 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4321 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4381 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     4441 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4501 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4561 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     4621 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4681 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4741 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4801 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     4861 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     4921 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     4981 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5041 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5101 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5161 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5221 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     5281 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     5341 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5401 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     5461 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     5521 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     5581 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     5641 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     5701 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     5761 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     5821 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     5881 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     5941 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6001 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6061 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6121 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6181 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     6241 ctggcctttt gctcacatgt
//
