LOCUS       sequence-457461-        6438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  produces AAV expressing Cal-light M13-TevC component and TdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6438)
  AUTHORS   Lee D, Hyun JH, Jung K, Hannan P, Kwon HB
  TITLE     A calcium- and light-gated switch to induce gene expression in 
            activated neurons.
  JOURNAL   Nat Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3902.
  PUBMED    28650460
REFERENCE   2  (bases 1 to 6438)
  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 
            Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3902."
COMMENT     Sequence Label: pAAV-M13-TEV-C-P2A-TdTomato
FEATURES             Location/Qualifiers
     source          1..6438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..474
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     promoter        494..512
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     494..511
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             547..564
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             568..600
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(604..612,613..627)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      634..643
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             655..711
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     CDS             1075..1131
                     /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             1138..2568
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     misc_feature    2587..3175
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2640..2660)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3058..3069)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3216..3423
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3593..3722
                     /label=AAV2 ITR
     rep_origin      complement(3959..4547)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4039..4058)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4718..5509,5510..5578))
                     /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     5341..5360
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5579..5683)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5709..6164)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5846..5867)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6058..6077
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6236..6365
                     /label=AAV2 ITR
ORIGIN
        1 ctagactgca gagggccctg cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg
       61 ggtgggggtg cctacctgac gaccgacccc gacccactgg acaagcaccc aacccccatt
      121 ccccaaattg cgcatcccct atcagagagg gggaggggaa acaggatgcg gcgaggcgcg
      181 tgcgcactgc cagcttcagc accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc
      241 caccgccgcc tcagcactga aggcgcgctg acgtcactcg ccggtccccc gcaaactccc
      301 cttcccggcc accttggtcg cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg
      361 aggcgcgaga taggggggca cgggcgcgac catctgcgct gcggcgccgg cgactcagcg
      421 ctgcctcagt ctgcggtggg cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga
      481 ggatctaagc ttgatttagg tgacactata gaatacaagc tacttgttct ttttgcagga
      541 tccatgcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
      601 cgggatctgt acgacgatga cgataaggat ctcgccacca tggtcgactc atcacgtcgt
      661 aagtggaata agacaggtca cgcagtcaga gctataggtc ggctgagctc actcgaaggt
      721 ggcggtggct ctggaggtgg tgggtccgga ggaggcggca tcgagatgtc cagcatggtg
      781 agcgatacta gctgtacctt cccatcatct gacggaatct tctggaagca ctggattcag
      841 actaaggacg gccagtgtgg cagcccactg gtgagcacac gagacggatt catcgtgggg
      901 attcacagcg cctccaactt tacaaacacc aataactatt tcacctcagt gccaaagaac
      961 tttatggagc tgctgaccaa ccaggaggcc cagcagtggg tgagcgggtg gcgcctgaac
     1021 gccgattccg tgctgtgggg cgggcacaag gtgtttatgg catgcggaag cggagctact
     1081 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tgaattcatg
     1141 gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg catggagggc
     1201 tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1261 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
     1321 ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc
     1381 gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1441 gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac
     1501 aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc
     1561 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag
     1621 atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagaccatc
     1681 tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac
     1741 atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc
     1801 caccacctgt tcctggggca tggcaccggc agcaccggca gcggcagctc cggcaccgcc
     1861 tcctccgagg acaacaacat ggccgtcatc aaagagttca tgcgcttcaa ggtgcgcatg
     1921 gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     1981 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     2041 gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca ccccgccgac
     2101 atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     2161 ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga cggcacgctg
     2221 atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag
     2281 aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag
     2341 ggcgagatcc accaggccct gaagctgaag gacggcggcc gctacctggt ggagttcaag
     2401 accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt ggacaccaag
     2461 ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag
     2521 ggccgccacc acctgttcct gtacggcatg gacgagctgt acaagtagtc tagccagctt
     2581 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2641 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2701 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2761 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     2821 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     2881 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     2941 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3001 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3061 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3121 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3181 taccgtcgac ccgggcggcc gctcataaag ctcgactgtg ccttctagtt gccagccatc
     3241 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3301 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3361 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3421 ggatgcggtc cggactgtac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3481 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatcttc
     3541 ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc
     3601 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3661 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     3721 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3781 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3841 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3901 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3961 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4021 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4081 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4141 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4201 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4261 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4321 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4381 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4441 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4501 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4561 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4621 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4681 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4741 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4801 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4861 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4921 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4981 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5041 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5101 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5161 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5221 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5281 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5341 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5401 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5461 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5521 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5581 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5641 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5701 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5761 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5821 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5881 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5941 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     6001 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     6061 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     6121 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag
     6181 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgcg
     6241 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
     6301 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
     6361 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa
     6421 gatcgtaggt taattaat
//
