LOCUS       sequence-77369-e        7255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Forebrain principle neuron specific expression of GCaMP6f 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 7255)
  TITLE     Ting GCaMP6 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7255)
  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            "Unpublished"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-GCaMP6f-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        164..1452
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1465..1474
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1471..1479,1480..1497,1498..1500,1501..1533,1534..1536,
                     1537..1560,1561..1587,1588..1644,1645..1650,1651..1920,
                     1921..1938,1939..2373,2374..2379,2380..2820)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1567..1576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1857..1878
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1984..2005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2245..2264)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2836..2892
                     /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             2905..2925
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2926..3624
                     /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(3056..3074)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3324..3343
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3518..3541
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    3674..4262
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3727..3747)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4145..4156)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4295..4406
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4518..4658
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4518..4647
                     /label=AAV2 ITR
     rep_origin      4733..5188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4820..4839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5030..5051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5205..5224)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5324..5346
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5384..5402)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5470..5574
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5575..5643,5644..6435)
                     /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(5793..5812)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6606..7194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7095..7114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtttaat taacattatg gccttaggtc
      181 acttcatctc catggggttc ttcttctgat tttctagaaa atgagatggg ggtgcagaga
      241 gcttcctcag tgacctgccc agggtcacat cagaaatgtc agagctagaa cttgaactca
      301 gattactaat cttaaattcc atgccttggg ggcatgcaag tacgatatac agaaggagtg
      361 aactcattag ggcagatgac caatgagttt aggaaagaag agtccagggc agggtacatc
      421 tacaccaccc gcccagccct gggtgagtcc agccacgttc acctcattat agttgcctct
      481 ctccagtcct accttgacgg gaagcacaag cagaaactgg gacaggagcc ccaggagacc
      541 aaatcttcat ggtccctctg ggaggatggg tggggagagc tgtggcagag gcctcaggag
      601 gggccctgct gctcagtggt gacagatagg ggtgagaaag cagacagagt cattccgtca
      661 gcattctggg tctgtttggt acttcttctc acgctaaggt ggcggtgtga tatgcacaat
      721 ggctaaaaag cagggagagc tggaaagaaa caaggacaga gacagaggcc aagtcaacca
      781 gaccaattcc cagaggaagc aaagaaacca ttacagagac tacaaggggg aagggaagga
      841 gagatgaatt agcttcccct gtaaacctta gaacccagct gttgccaggg caacggggca
      901 atacctgtct cttcagagga gatgaagttg ccagggtaac tacatcctgt ctttctcaag
      961 gaccatccca gaatgtggca cccactagcc gttaccatag caactgcctc tttgccccac
     1021 ttaatcccat cccgtctgtt aaaagggccc tatagttgga ggtgggggag gtaggaagag
     1081 cgatgatcac ttgtggacta agtttgttcg catccccttc tccaaccccc tcagtacatc
     1141 accctggggg aacagggtcc acttgctcct gggcccacac agtcctgcag tattgtgtat
     1201 ataaggccag ggcaaagagg agcaggtttt aaagtgaaag gcaggcaggt gttggggagg
     1261 cagttaccgg ggcaacggga acagggcgtt tcggaggtgg ttgccatggg gacctggatg
     1321 ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc tgctcagaag ccccaagctc
     1381 gtcagtcaag ccggttctcc gtttgcactc aggagcacgg gcaggcgagt ggcccctagt
     1441 tctgggggca gcgggggatc cggcgccacc atgggttctc atcatcatca tcatcatggt
     1501 atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag
     1561 gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc
     1621 agagctatag gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag
     1681 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     1741 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1801 cactacctga gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg
     1861 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1921 ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1981 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt
     2041 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2101 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2161 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc
     2221 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2281 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2341 ggcaacatcc tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag
     2401 atcgcagaat ttaaagagga attctcccta tttgacaagg acggggatgg gacaataaca
     2461 accaaggagc tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg
     2521 caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc
     2581 ctgacaatga tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg
     2641 ttcggtgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg
     2701 atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca
     2761 gacatcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag
     2821 gataacggaa gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag
     2881 aatcccgggc catctagaat ggcccccaag aagaagagga aggtggtgag caagggcgag
     2941 gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac
     3001 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     3061 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
     3121 atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg
     3181 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg
     3241 accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc
     3301 ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     3361 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg
     3421 aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag
     3481 cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac
     3541 gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg
     3601 tacggcatgg acgagctgta caagtaagtc gacggcgcgc ccctgcaggg aattcgatat
     3661 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3721 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3781 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3841 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3901 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3961 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     4021 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     4081 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     4141 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     4201 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4261 gcatcgatac cgagcgctgc tcgagagatc tcgactgtgc cttctagttg ccagccatct
     4321 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4381 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4441 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgcacgt
     4501 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4561 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4621 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4681 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4741 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4801 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4861 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4921 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4981 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5041 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     5101 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5161 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5221 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5281 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5341 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5401 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5461 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5521 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5581 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5641 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5701 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5761 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5821 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5881 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5941 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6001 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6061 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6121 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6181 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6241 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6301 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6361 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6421 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6481 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6541 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6601 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6661 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6721 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     6781 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6841 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6901 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6961 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7021 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7081 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7141 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7201 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
