LOCUS       sequence-172966-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuronal 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 6739)
  TITLE     Ting GCaMP6 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6739)
  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            "Unpublished"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-hSyn1-GCaMP6s-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   405..534
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(596..1184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(676..695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1355..2146,2147..2215))
                     /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     1978..1997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2216..2320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2388..2406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2444..2466)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2566..2585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2602..3057)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2739..2760)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2951..2970
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3132..3272
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3143..3272
                     /label=AAV2 ITR
     polyA_signal    3312..3788
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     3570..3589
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    3820..4408
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3926..3937
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4335..4355
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4458..5156)
                     /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(4541..4564)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4739..4758)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5008..5026
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5157..5177)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5190..5246)
                     /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(5262..5702,5703..5708,5709..6143,6144..6161,
                     6162..6431,6432..6437,6438..6494,6495..6521,6522..6545,
                     6546..6548,6549..6581,6582..6584,6585..6602,6603..6611))
                     /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     5818..5837
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6077..6098
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6204..6225)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      6506..6515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      6608..6617
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(6644..6739,1..352))
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 cccccctatc tcgcgcctcg cgtggtgcgg tccggctggg ccggcggcgg cgcggacgcg
       61 accaaggtgg ccgggaaggg gagtttgcgg gggaccggcg agtgacgtca gcgcgccttc
      121 agtgctgagg cggcggtggc gcgcgccgcc aggcgggggc gaaggcactg tccgcggtgc
      181 tgaagctggc agtgcgcacg cgcctcgccg catcctgttt cccctccccc tctctgatag
      241 gggatgcgca atttggggaa tgggggttgg gtgcttgtcc agtgggtcgg ggtcggtcgt
      301 caggtaggca cccccacccc gcctcatcct ggtcctaaaa cccacttgca ctcatacgca
      361 gggccctctg cagtctagac acttaattaa acgcgtgcgg ccgcaggaac ccctagtgat
      421 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
      481 cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggacatgt
      541 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      601 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      661 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      721 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      781 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
      841 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
      901 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
      961 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1021 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1081 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1141 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1201 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1261 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1321 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1381 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1441 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1501 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1561 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1621 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     1681 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1741 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1801 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     1861 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     1921 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     1981 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2041 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2101 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2161 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2221 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     2281 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2341 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     2401 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     2461 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     2521 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     2581 ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa
     2641 atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa atcccttata
     2701 aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac aagagtccac
     2761 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2821 cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2881 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2941 cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg
     3001 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtact
     3061 atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     3121 catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa
     3181 gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga
     3241 gggagtggcc aactccatca ctaggggttc ctgcggccgc tcggtccgca cgtggttacc
     3301 tacaaaatca gaaggacagg gaagggagca gtggttcacg cctgtaatcc cagcaatttg
     3361 ggaggccaag gtgggtagat cacctgagat taggagttgg agaccagcct ggccaatatg
     3421 gtgaaacccc gtctctacca aaaaaacaaa aattagctga gcctggtcat gcatgcctgg
     3481 aatcccaaca actcgggagg ctgaggcagg agaatcgctt gaacccagga ggcggagatt
     3541 gcagtgagcc aagattgtgc cactgcactc cagcttggtt cccaatagac cccgcaggcc
     3601 ctacaggttg tcttcccaac ttgccccttg ctccatacca cccccctcca ccccataata
     3661 ttatagaagg acacctagtc agacaaaatg atgcaactta attttattag gacaaggctg
     3721 gtgggcactg gagtggcaac ttccagggcc aggagaggca ctggggaggg gtcacaggga
     3781 tgccacccgt agatctctcg agcagcgctc ggtatcgatg cggggaggcg gcccaaaggg
     3841 agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca
     3901 ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag aaggacgtcc
     3961 cgcgcagaat ccaggtggca acataggcga gcagccaagg aaaggacgat gatttccccg
     4021 acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc
     4081 aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc ccggaaagga
     4141 gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac acagtgcaca
     4201 ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc aaccaggatt
     4261 tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta
     4321 aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt
     4381 cacaaatttt gtaatccaga ggttgattat cgataagctt gatatcgaat tccctgcagg
     4441 ggcgcgccgt cgacttactt gtacagctcg tccatgccgt acaggaacag gtggtggcgg
     4501 ccctcggagc gctcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     4561 agcttggtgt ccacgtagta gtagccgggc agttgcacgg gcttcttggc catgtagatg
     4621 gtcttgaact ccaccaggta gtggccgccg tccttcagct tcagggcctg gtggatctcg
     4681 cccttcagca cgccgtcgcg ggggtacagg cgctcggtgg aggcctccca gcccatggtc
     4741 ttcttctgca ttacggggcc gtcggggggg aagttggtgc cgcgcatctt caccttgtag
     4801 atcagcgtgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccagacc gccgtcctcg
     4861 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttctt gtaatcgggg
     4921 atgtcggcgg ggtgcttcac gtacgccttg gagccgtaca tgaactgggg ggacaggatg
     4981 tcccaggcga agggcagggg gccgcccttg gtcaccttca gcttggcggt ctgggtgccc
     5041 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt catggagccc
     5101 tccatgcgca ccttgaagcg catgaactct ttgatgacct cctcgccctt gctcaccacc
     5161 ttcctcttct tcttgggggc cattctagat ggcccgggat tctcttcgac atcccctgct
     5221 tgtttcaaca gggagaagtt agtggctccg cttccgttat ccttcgctgt catcatttgt
     5281 acaaactctt cgtagtttac ctgaccatcc ccatcgatgt ctgcttccct gatcatttca
     5341 tcaacctctt catctgttaa cttctctcca aggtttgtca tcacgtggcg aagctctgct
     5401 gcactgatgt agccattgcc atccttatca aacacaccga acgcttctct aatttcttct
     5461 tccgtgtccc tgtatttcat ttttcttgcc atcattgtca ggaactcagg gaagtcgatt
     5521 gtgccgtcac cgtcggcatc tacttcattg atcatgtcct gcagctctgc ttctgtgggg
     5581 ttctgcccca gagaccgcat caccgtcccc agctccttgg ttgttattgt cccatccccg
     5641 tccttgtcaa atagggagaa agcctcttta aattctgcga tctgctcttc agtcagttgg
     5701 tccggcaggt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
     5761 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
     5821 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggatgtagcc ttcgggcatg
     5881 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
     5941 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     6001 atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc ggacacgctg
     6061 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     6121 agctcctcgc ccttgctcac catgctccct ccggtaccgc ccttgtacag ctcgtccatg
     6181 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
     6241 ttggggtctt tcgaaagttt ggactgcacg ctcaggtagt ggttgtcggg cagcagcacg
     6301 gggccgtcgc cgatgggggt gttctgctgg tagtggtagg cgagctgcac gccgccgtcc
     6361 tcgatgttgt ggcggatgtg gaagttcgcc ttgatgccgt tcttctgctt gtcggccttg
     6421 atatagacgt tctcgagtga gctcagccga cctatagctc tgactgcgtg acctgtctta
     6481 ttccacttac gacgtgatga gtcgaccatg gtggcgagat ccttatcgtc atcgtcgtac
     6541 agatcccgac ccatttgctg tccaccagtc atgctagcca taccatgatg atgatgatga
     6601 tgagaaccca tggtggcgcc ggatcctcta gccggtttct cgactgcgct ctcaggcacg
     6661 acacgactcc tccgctgccc accgcagact gaggcagcgc tgagtcgccg gcgccgcagc
     6721 gcagatggtc gcgcccgtg
//
