LOCUS       sequence-182952-        6638 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6638)
  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 6638)
  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..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR
     promoter        442..718
                     /label=chicken beta-actin promoter
     primer_bind     997..1019
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1081..1100
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1134..1167)
                     /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(1218..1251)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2609..2628
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2868..2889
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2931..2940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2953..2986
                     /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    3037..3070
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3083..3671
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3136..3156)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3554..3565)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3714..3835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3725..3744)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3779..3798
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3997..4126
                     /label=AAV2 ITR
     rep_origin      complement(4363..4951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4443..4462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5122..5913,5914..5982))
                     /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     5745..5764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5983..6087)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6113..6568)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6250..6271)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6462..6481
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt agccatgctc
      181 taggaagagt accattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      241 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      301 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      361 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      421 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      481 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      541 tgggggcggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg
      601 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
      661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
      721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
      781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
      901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
      961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat cttataactt
     1141 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
     1201 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tactagttga
     1261 tcatcaatcc tcaatgttgt gacggatctt aaaattagcc ttaatgccgt tcttttgctt
     1321 atcggctgtg atgtaaacat tgtgactgtt gtagttgtat tccaacttat gcccgagtat
     1381 gtttccgtcc tccttaaagt cgattccctt cagctcgatt cggttgacaa gtgtatcccc
     1441 ttcgaatttc acctcggccc tagtcttgta attaccatcg tccttgaaga agatagttct
     1501 ctcctggacg tacccctctg gcatggctga tttaaagaaa tcgtgttgtt tcatatggtc
     1561 tgggtagcgg gcgaaacaca taaggccata tcctagagta gtaacgagtg tgggccatgg
     1621 cacgggcagt ttccccgttg tgcagatgag cttgagtgtc aatttgccat tagttgcgtc
     1681 cccctcaccc tcccctcgca cgctgaattt atgaccgttg acatccccat ccagttccac
     1741 caaaataggg actacaccag tgaacaattc ttcgcccttg ctcaccatag atcccccggt
     1801 cccacctttg tacagctcgt ccattccgag agtgattccg gcggcggtga caaattccag
     1861 caggaccatg tgatcccgct tttcattagg atctttggaa agcttggact ggtatgacag
     1921 gtagtggttg tcaggcagga gaaccggccc atccccgatc ggagtattct gctggtagtg
     1981 atctgcaagc tggaccccac ccatgagctc ggggtagatg ggctgcacgt tttccaccat
     2041 cttcaggaac tcgtcgaaat cgattctgcc gttcttgtcg gtgtcggagt cgccgaagat
     2101 ctcgtccacg tcctcgtcgg tcagctgctc gccggtcagt ctgatgatgt cgccaaactc
     2161 ctctctgtcg atgaagccgt tgccgtcgaa gtcgaagatt ctgaagcact cgctcagttc
     2221 ctcttcgctg gcgtcggcaa cttgcatgcg ggcggcggtc acgaactcca gcaggaccat
     2281 gtgatcgcgc ttctcgttgg ggtctttgct cagcttggac tgggtgctca ggtagtggtt
     2341 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     2401 ctgcacgctg ccgtcctcga tgttgcagtt gaggccgaag ttggccttga tgccgttctt
     2461 ctgcttgtcg gcggtgatat agacgttgcc gtggatggcg ttgtactcca gcttgtgccc
     2521 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     2581 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2641 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2701 gtggtcgggg tagcgggcga agcactgcac gccccagctc agggtggtca cgagggtggg
     2761 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2821 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2881 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc
     2941 gctagcctcg agataacttc gtataaagta tcctatacga agttatttgc cttaacccag
     3001 aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa tgtatgctat
     3061 acgaagttat aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     3121 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3181 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3241 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     3301 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     3361 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     3421 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     3481 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     3541 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     3601 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3661 cgcctccccg catcgatacc gtcgacccgg gcggccgctt cgagcagaca tgataagata
     3721 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     3781 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     3841 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag
     3901 caagtaaaac ctctacaaat gtggtaaaat cgataaggat cttcctagag catggctacg
     3961 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     4021 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4081 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc attaatgaat
     4141 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4201 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4261 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4321 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4381 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4441 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4501 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4561 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4621 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4681 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4741 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4801 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4861 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4921 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4981 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5041 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5101 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5161 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5221 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5281 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5341 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5401 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5461 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5521 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5581 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5641 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5701 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5761 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5821 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5881 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5941 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6001 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6061 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     6121 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     6181 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     6241 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     6301 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     6361 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     6421 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     6481 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     6541 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg
     6601 gaagggcgat cggtgcgggc ctcttcgcta ttacgcca
//
