LOCUS       sequence-148706-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuronal 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 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  JOURNAL   Unpublished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            "Unpublished"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-GCaMP6f-P2A-nls-dTomato
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..468
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      495..504
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(501..509,510..527,528..530,531..563,564..566,567..590,
                     591..617,618..674,675..680,681..950,951..968,969..1403,
                     1404..1409,1410..1850)
                     /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      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     887..908
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1014..1035)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1275..1294)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1866..1922
                     /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             1935..1955
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1956..2654
                     /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(2086..2104)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2354..2373
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2548..2571
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    2704..3292
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2757..2777)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3175..3186)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3324..3800
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3523..3542)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3840..3980
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3840..3969
                     /label=AAV2 ITR
     rep_origin      4055..4510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4142..4161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4352..4373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4527..4546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4646..4668
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4706..4724)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4792..4896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4897..4965,4966..5757)
                     /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(5115..5134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5928..6516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6417..6436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6578..6707
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag gcggggtggg
       61 ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa
      121 attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca
      181 ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc
      241 cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc
      301 ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc
      361 gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct
      421 cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gagaaaccgg
      481 ctagaggatc cggcgccacc atgggttctc atcatcatca tcatcatggt atggctagca
      541 tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatctcgcca
      601 ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc agagctatag
      661 gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag aacggcatca
      721 aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact
      781 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      841 gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      901 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
      961 gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1021 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg
     1081 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1141 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1201 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
     1261 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1321 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1381 tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag atcgcagaat
     1441 ttaaagagga attctcccta tttgacaagg acggggatgg gacaataaca accaaggagc
     1501 tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg caggacatga
     1561 tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc ctgacaatga
     1621 tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg ttcggtgtgt
     1681 ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg atgacaaacc
     1741 ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca gacatcgatg
     1801 gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag gataacggaa
     1861 gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
     1921 catctagaat ggcccccaag aagaagagga aggtggtgag caagggcgag gaggtcatca
     1981 aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga
     2041 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     2101 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct
     2161 ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg
     2221 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc
     2281 aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact
     2341 tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc
     2401 gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg
     2461 acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag cccgtgcaac
     2521 tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca
     2581 ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg tacggcatgg
     2641 acgagctgta caagtaagtc gacggcgcgc ccctgcaggg aattcgatat caagcttatc
     2701 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     2761 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     2821 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     2881 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     2941 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3001 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3061 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     3121 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3181 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     3241 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     3301 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
     3361 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
     3421 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
     3481 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
     3541 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
     3601 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
     3661 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
     3721 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
     3781 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
     3841 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     3901 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     3961 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4021 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     4081 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     4141 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4201 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4261 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4321 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4381 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     4441 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4501 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4561 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4621 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4681 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4741 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4801 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4861 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4921 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4981 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5041 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5101 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5161 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5221 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5281 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5341 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5401 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5461 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5521 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5581 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5641 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5701 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5761 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5821 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5881 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5941 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6001 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6061 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6121 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6181 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6241 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6301 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6361 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6421 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6481 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6541 cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc
     6601 tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
     6661 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg
     6721 tttaattaag tgtctagac
//
