LOCUS       sequence-469612-        7251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Forebrain principle neuron specific expression of GCaMP6s Ca sensor 
            and physically separate nuclear localized dTomato fluorophore .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7251)
  TITLE     Ting GCaMP6 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7251)
  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: 
            "Unpublished"
COMMENT     Sequence Label: AAV-CaMKIIa-GCaMP6s-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..7251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(168..187)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     437..455
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(586..606)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(619..675)
                     /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             complement(join(691..1131,1132..1137,1138..1572,1573..1590,
                     1591..1860,1861..1866,1867..1923,1924..1950,1951..1974,
                     1975..1977,1978..2010,2011..2013,2014..2031,2032..2040))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     1247..1266
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1506..1527
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1633..1654)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1935..1944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2037..2046
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2059..3347)
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     repeat_region   3370..3499
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3561..4149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3641..3660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4320..5111,5112..5180))
                     /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     4943..4962
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5181..5285)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5353..5371
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5409..5431)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5531..5550
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5567..6022)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5704..5725)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5916..5935
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6097..6237
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6108..6237
                     /label=AAV2 ITR
     polyA_signal    6355..6466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    6500..7088
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6606..6617
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7015..7035
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7138..7251,1..585))
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLYGMDELYK"
     primer_bind     complement(7221..7244)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
ORIGIN
        1 cacgtagtag tagccgggca gttgcacggg cttcttggcc atgtagatgg tcttgaactc
       61 caccaggtag tggccgccgt ccttcagctt cagggcctgg tggatctcgc ccttcagcac
      121 gccgtcgcgg gggtacaggc gctcggtgga ggcctcccag cccatggtct tcttctgcat
      181 tacggggccg tcggggggga agttggtgcc gcgcatcttc accttgtaga tcagcgtgcc
      241 gtcctgcagg gaggagtcct gggtcacggt caccagaccg ccgtcctcga agttcatcac
      301 gcgctcccac ttgaagccct cggggaagga cagcttcttg taatcgggga tgtcggcggg
      361 gtgcttcacg tacgccttgg agccgtacat gaactggggg gacaggatgt cccaggcgaa
      421 gggcaggggg ccgcccttgg tcaccttcag cttggcggtc tgggtgccct cgtaggggcg
      481 gccctcgccc tcgccctcga tctcgaactc gtggccgttc atggagccct ccatgcgcac
      541 cttgaagcgc atgaactctt tgatgacctc ctcgcccttg ctcaccacct tcctcttctt
      601 cttgggggcc attctagatg gcccgggatt ctcttcgaca tcccctgctt gtttcaacag
      661 ggagaagtta gtggctccgc ttccgttatc cttcgctgtc atcatttgta caaactcttc
      721 gtagtttacc tgaccatccc catcgatgtc tgcttccctg atcatttcat caacctcttc
      781 atctgttaac ttctctccaa ggtttgtcat cacgtggcga agctctgctg cactgatgta
      841 gccattgcca tccttatcaa acacaccgaa cgcttctcta atttcttctt ccgtgtccct
      901 gtatttcatt tttcttgcca tcattgtcag gaactcaggg aagtcgattg tgccgtcacc
      961 gtcggcatct acttcattga tcatgtcctg cagctctgct tctgtggggt tctgccccag
     1021 agaccgcatc accgtcccca gctccttggt tgttattgtc ccatccccgt ccttgtcaaa
     1081 tagggagaaa gcctctttaa attctgcgat ctgctcttca gtcagttggt ccggcaggtt
     1141 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     1201 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     1261 gtcgtccttg aagaagatgg tgcgctcctg gatgtagcct tcgggcatgg cggacttgaa
     1321 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
     1381 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     1441 ggtcagcttg ccgtaggtgg catcgccctc accctcgccg gacacgctga acttgtggcc
     1501 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     1561 cttgctcacc atgctccctc cggtaccgcc cttgtacagc tcgtccatgc cgagagtgat
     1621 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     1681 cgaaagtttg gactgcacgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     1741 gatgggggtg ttctgctggt agtggtaggc gagctgcacg ccgccgtcct cgatgttgtg
     1801 gcggatgtgg aagttcgcct tgatgccgtt cttctgcttg tcggccttga tatagacgtt
     1861 ctcgagtgag ctcagccgac ctatagctct gactgcgtga cctgtcttat tccacttacg
     1921 acgtgatgag tcgaccatgg tggcgagatc cttatcgtca tcgtcgtaca gatcccgacc
     1981 catttgctgt ccaccagtca tgctagccat accatgatga tgatgatgat gagaacccat
     2041 ggtggcgccg gatcccccgc tgcccccaga actaggggcc actcgcctgc ccgtgctcct
     2101 gagtgcaaac ggagaaccgg cttgactgac gagcttgggg cttctgagca gggcactgtg
     2161 gctgctcaca gcctcgcgag ccttcgtcag catccaggtc cccatggcaa ccacctccga
     2221 aacgccctgt tcccgttgcc ccggtaactg cctccccaac acctgcctgc ctttcacttt
     2281 aaaacctgct cctctttgcc ctggccttat atacacaata ctgcaggact gtgtgggccc
     2341 aggagcaagt ggaccctgtt cccccagggt gatgtactga gggggttgga gaaggggatg
     2401 cgaacaaact tagtccacaa gtgatcatcg ctcttcctac ctcccccacc tccaactata
     2461 gggccctttt aacagacggg atgggattaa gtggggcaaa gaggcagttg ctatggtaac
     2521 ggctagtggg tgccacattc tgggatggtc cttgagaaag acaggatgta gttaccctgg
     2581 caacttcatc tcctctgaag agacaggtat tgccccgttg ccctggcaac agctgggttc
     2641 taaggtttac aggggaagct aattcatctc tccttccctt cccccttgta gtctctgtaa
     2701 tggtttcttt gcttcctctg ggaattggtc tggttgactt ggcctctgtc tctgtccttg
     2761 tttctttcca gctctccctg ctttttagcc attgtgcata tcacaccgcc accttagcgt
     2821 gagaagaagt accaaacaga cccagaatgc tgacggaatg actctgtctg ctttctcacc
     2881 cctatctgtc accactgagc agcagggccc ctcctgaggc ctctgccaca gctctcccca
     2941 cccatcctcc cagagggacc atgaagattt ggtctcctgg ggctcctgtc ccagtttctg
     3001 cttgtgcttc ccgtcaaggt aggactggag agaggcaact ataatgaggt gaacgtggct
     3061 ggactcaccc agggctgggc gggtggtgta gatgtaccct gccctggact cttctttcct
     3121 aaactcattg gtcatctgcc ctaatgagtt cactccttct gtatatcgta cttgcatgcc
     3181 cccaaggcat ggaatttaag attagtaatc tgagttcaag ttctagctct gacatttctg
     3241 atgtgaccct gggcaggtca ctgaggaagc tctctgcacc cccatctcat tttctagaaa
     3301 atcagaagaa gaaccccatg gagatgaagt gacctaaggc cataatgtta attaaacgcg
     3361 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3421 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg cggcctcagt gagcgagcga
     3481 gcgcgcagct gcctgcagga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3541 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3601 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3661 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3721 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3781 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3841 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3901 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3961 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4021 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4081 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4141 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4201 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4261 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4321 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4381 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4441 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4501 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4561 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4621 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4681 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4741 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4801 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4861 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4921 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4981 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5041 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5101 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5161 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5221 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5281 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5341 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     5401 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     5461 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     5521 cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaaat tgtaaacgtt
     5581 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5641 accgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagataga gttgagtgtt
     5701 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5761 aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc aagttttttg
     5821 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5881 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5941 gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     6001 aatgcgccgc tacagggcgc gtactatggt tgctttgacg tatgcggtgt gaaataccgc
     6061 acagatgcgt aaggagaaaa taccgcatca ggcgcccctg caggcagctg cgcgctcgct
     6121 cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct
     6181 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg
     6241 gccgctcggt ccgcacgtga gatctcatgc ctgctattgt cttcccaatc ctcccccttg
     6301 ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca
     6361 atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc
     6421 acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga agatctctcg
     6481 agcagcgctc ggtatcgatg cggggaggcg gcccaaaggg agatccgact cgtctgaggg
     6541 cgaaggcgaa gacgcggaag aggccgcaga gccggcagca ggccgcggga aggaaggtcc
     6601 gctggattga gggccgaagg gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca
     6661 acataggcga gcagccaagg aaaggacgat gatttccccg acaacaccac ggaattgtca
     6721 gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc aggcggcgat gagttccgcc
     6781 gtggcaatag ggagggggaa agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg
     6841 ccccaaccag tgggggttgc gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca
     6901 acgggccaca actcctcata aagagacagc aaccaggatt tatacaagga ggagaaaatg
     6961 aaagccatac gggaagcaat agcatgatac aaaggcatta aagcagcgta tccacatagc
     7021 gtaaaaggag caacatagtt aagaatacca gtcaatcttt cacaaatttt gtaatccaga
     7081 ggttgattat cgataagctt gatatcgaat tccctgcagg ggcgcgccgt cgacttactt
     7141 gtacagctcg tccatgccgt acaggaacag gtggtggcgg ccctcggagc gctcgtactg
     7201 ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc agcttggtgt c
//
