LOCUS       sequence-193782-        6605 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  AAV expression of FLEXed Twitch2B driven by CAG promoter. Twitch 2B 
            is a fluorescent reporter for calcium signaling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6605)
  AUTHORS   Thestrup T, Litzlbauer J, Bartholomaus I, Mues M, Russo L, Dana H, 
            Kovalchuk Y, Liang Y, Kalamakis G, Laukat Y, Becker S, Witte G, 
            Geiger A, Allen T, Rome LC, Chen TW, Kim DS, Garaschuk O, Griesinger
            C, Griesbeck O
  TITLE     Optimized ratiometric calcium sensors for functional in vivo imaging
            of neurons and T lymphocytes.
  JOURNAL   Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773.
  PUBMED    24390440
REFERENCE   2  (bases 1 to 6605)
  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: "Nat 
            Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773."
COMMENT     Sequence Label: pAAV.CAG.Flex.Twitch2B.WPRE.SV40
FEATURES             Location/Qualifiers
     source          1..6605
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     406..428
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     490..509
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(543..576)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(627..660)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2018..2037
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2277..2298
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2340..2349
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2362..2395
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2446..2479
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2492..3080
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2545..2565)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2963..2974)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3123..3244
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3134..3153)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3188..3207
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3406..3535
                     /label=AAV2 ITR
     rep_origin      complement(3772..4360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3852..3871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4531..5322,5323..5391))
                     /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     5154..5173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5392..5496)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5522..5977)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5659..5680)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5871..5890
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6049..6178
                     /label=AAV2 ITR
ORIGIN
        1 ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg
       61 aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg
      121 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
      181 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      241 ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
      301 gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcggggct
      361 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
      421 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
      481 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
      541 ttataacttc gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc
      601 tgctattagc aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat
      661 actagttgat catcaatcct caatgttgtg acggatctta aaattagcct taatgccgtt
      721 cttttgctta tcggctgtga tgtaaacatt gtgactgttg tagttgtatt ccaacttatg
      781 cccgagtatg tttccgtcct ccttaaagtc gattcccttc agctcgattc ggttgacaag
      841 tgtatcccct tcgaatttca cctcggccct agtcttgtaa ttaccatcgt ccttgaagaa
      901 gatagttctc tcctggacgt acccctctgg catggctgat ttaaagaaat cgtgttgttt
      961 catatggtct gggtagcggg cgaaacacat aaggccatat cctagagtag taacgagtgt
     1021 gggccatggc acgggcagtt tccccgttgt gcagatgagc ttgagtgtca atttgccatt
     1081 agttgcgtcc ccctcaccct cccctcgcac gctgaattta tgaccgttga catccccatc
     1141 cagttccacc aaaataggga ctacaccagt gaacaattct tcgcccttgc tcaccataga
     1201 tcccccggtc ccacctttgt acagctcgtc cattccgaga gtgattccgg cggcggtgac
     1261 aaattccagc aggaccatgt gatcccgctt ttcattagga tctttggaaa gcttggactg
     1321 gtatgacagg tagtggttgt caggcaggag aaccggccca tccccgatcg gagtattctg
     1381 ctggtagtga tctgcaagct ggaccccacc catgagctcg gggtagatgg gctgcacgtt
     1441 ttccaccatc ttcaggaact cgtcgaaatc gattctgccg ttcttgtcgg tgtcggagtc
     1501 gccgaagatc tcgtccacgt cctcgtcggt cagctgctcg ccggtcagtc tgatgatgtc
     1561 gccaaactcc tctctgtcga tgaagccgtt gccgtcgaag tcgaagattc tgaagcactc
     1621 gctcagttcc tcttcgctgg cgtcggcaac ttgcatgcgg gcggcggtca cgaactccag
     1681 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agcttggact gggtgctcag
     1741 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
     1801 gtcggcgagc tgcacgctgc cgtcctcgat gttgcagttg aggccgaagt tggccttgat
     1861 gccgttcttc tgcttgtcgg cggtgatata gacgttgccg tggatggcgt tgtactccag
     1921 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
     1981 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
     2041 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
     2101 ctgcttcatg tggtcggggt agcgggcgaa gcactgcacg ccccagctca gggtggtcac
     2161 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     2221 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     2281 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     2341 catggtggcg ctagcctcga gataacttcg tataaagtat cctatacgaa gttatttgcc
     2401 ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat
     2461 gtatgctata cgaagttata agcttatcga taatcaacct ctggattaca aaatttgtga
     2521 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     2581 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     2641 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     2701 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     2761 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     2821 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     2881 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     2941 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3001 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3061 cctttgggcc gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat
     3121 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3181 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3241 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     3301 tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc
     3361 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     3421 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3481 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca
     3541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4381 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4441 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4501 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4561 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4621 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4681 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4741 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4801 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4861 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4921 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4981 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5041 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5101 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5161 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5221 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5281 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5341 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5401 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5461 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5521 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5581 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5641 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5701 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5761 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5821 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5881 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5941 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     6001 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc
     6061 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc
     6121 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg
     6181 tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag gaagagtacc
     6241 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     6301 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     6361 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     6421 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     6481 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     6541 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     6601 ggggg
//
