LOCUS       sequence-207281-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In the presence of Cre, drives neuron-specific expression of both 
            GCaMP6s fused to the presynaptic protein synaptophysin and 
            independently the red-fluorescent protein mRuby3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7232)
  TITLE     AAV GCaMP6 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7232)
  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            "Unpublished"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_CWSL.hSyn.DIO.Synaptophysin-GCaMP6s.P2A.mR...
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        170..617
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(661..694)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(745..778)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(782..1489)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     CDS             complement(1490..1546)
                     /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(1556..1996,1997..2002,2003..2437,2438..2455,
                     2456..2725,2726..2731,2732..2788,2789..2815,2816..2839,
                     2840..2842,2843..2875,2876..2878,2879..2896,2897..2905))
                     /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     2112..2131
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2371..2392
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2498..2519)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2800..2809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(2916..2930,2931..2939))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    3911..3944
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3995..4028
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4115..4135)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4495..4635
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4495..4624
                     /label=AAV2 ITR
     rep_origin      4710..5165
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4797..4816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5007..5028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5182..5201)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5301..5323
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5361..5379)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5447..5551
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5552..5620,5621..6412)
                     /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(5770..5789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6583..7171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7072..7091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcag atgctgcact gcagagggcc ctgcgtatga gtgcaagtgg
      181 gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac cccgacccac
      241 tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg
      301 gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt
      361 cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac
      421 tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg
      481 gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc
      541 gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt
      601 cgtgcctgag agcgcagacc ggttcgagaa ggtaccggat cctctagagt cgactccgga
      661 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
      721 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttattc
      781 acttgtacag ctcgtccatg ccaccaccaa ggttgctgta tttggcaact gccacttctc
      841 tttgcactac gtaggtttca ttgtcactct cctcgatcct ttccaggcgg tgatcaacgg
      901 catggacacc gggcatcttg atgttcccga cggtcttttt tgacctgtaa gttgtcacga
      961 agttgcagtg cagatggcca ccaccatcaa ctttcagtgc gatgtcagtg tatcctctca
     1021 gaccaccatc tgctggatac atcatctctg tattaggctc ccaacccttg gtcttcttct
     1081 gcatcacggg accattggag ggaaagttta cccctctgac cttgacgttg tagacgagct
     1141 cgccatcctc aaggctggtg tcctgcgtga cggtgacgac tccaccatct tcgtatctcg
     1201 taactctttc ccaagtaaaa ccctcaggaa aggactgttt aaagaaatca gggatgtcgg
     1261 ccgggtactt gataaaggta cggctgccat acatgaacga cgtggcaaga atgtcaaagg
     1321 caaatggcag gggtcctccc tcgatgactt tgatcctcat ggtttgcact ccctcgtacg
     1381 gtctgccttc tccttcacct gtgcatttga attggtggcc gttgaccgaa ccttccatga
     1441 ccaccttcat acgcatattt tccttgatca gctcttcgcc cttagacact ggcccgggat
     1501 tctcttcgac atcccctgct tgtttcaaca gggagaagtt agtggctccg cttcccttcg
     1561 ctgtcatcat ttgtacaaac tcttcgtagt ttacctgacc atccccatcg atgtctgctt
     1621 ccctgatcat ttcatcaacc tcttcatctg ttaacttctc tccaaggttt gtcatcacgt
     1681 ggcgaagctc tgctgcactg atgtagccat tgccatcctt atcaaacaca ccgaacgctt
     1741 ctctaatttc ttcttccgtg tccctgtatt tcatttttct tgccatcatt gtcaggaact
     1801 cagggaagtc gattgtgccg tcaccgtcgg catctacttc attgatcatg tcctgcagct
     1861 ctgcttctgt ggggttctgc cccagagacc gcatcaccgt ccccagctcc ttggttgtta
     1921 ttgtcccatc cccgtccttg tcaaataggg agaaagcctc tttaaattct gcgatctgct
     1981 cttcagtcag ttggtccggc aggttgtact ccagcttgtg ccccaggatg ttgccgtcct
     2041 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     2101 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggatgt
     2161 agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc
     2221 tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct
     2281 tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcaccct
     2341 cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca
     2401 ccaccccggt gaacagctcc tcgcccttgc tcaccatgct ccctccggta ccgcccttgt
     2461 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     2521 gatcgcgctt ctcgttgggg tctttcgaaa gtttggactg cacgctcagg tagtggttgt
     2581 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg taggcgagct
     2641 gcacgccgcc gtcctcgatg ttgtggcgga tgtggaagtt cgccttgatg ccgttcttct
     2701 gcttgtcggc cttgatatag acgttctcga gtgagctcag ccgacctata gctctgactg
     2761 cgtgacctgt cttattccac ttacgacgtg atgagtcgac catggtggcg agatccttat
     2821 cgtcatcgtc gtacagatcc cgacccattt gctgtccacc agtcatgcta gccataccat
     2881 gatgatgatg atgatgagaa cccattggcg agatccttat cgtcatcgtc gtacagatcc
     2941 cgacccattt gctgtccacc agtcccggtg gatcctgatt ggagaaggag gtgggcgcac
     3001 cctgttggcc atagccttgc tgcccatagt cgccctgagg cccgtagcca ccgccaccgc
     3061 tggctggctg cccgtaatcg ggttgataac caccctgagg cccgtaggag tcctggggcc
     3121 catagcctcc ggggccctgc ccgtagcccg catcgccgta ggcatcgcca ggtgctggtt
     3181 gcttttccgg ggcgcctgga ggtgcgcgca tgaatggggc tgcccagcct gtctccttga
     3241 acacgaacca taagttgcca acccagagca ccaggttcag gaagccaaac accactgtgg
     3301 tgttgagtcc tgaagtcaca gggtccctca gttccttgca tgtgttccct gtctggcggc
     3361 acatgggcat ctccttgata atgttctctg ggtccgtggc catcttcaca tcggacaggc
     3421 ctttggccca ggctgatgaa ctaactagcc acatgaaagc gaacacggct gtagccagaa
     3481 agtccatcat aggccctttg ttgttctctc ggtacttgtt ctgcaggaag atgtaggtgg
     3541 ccagggcccc catggagtag aggaaggcaa acacagccac ggtgacaaag aattcagccg
     3601 acgaggagta gtccccaacc aggaagatct tggtagtgcc ccctttgacg caggagggtg
     3661 catcaaagta cacttggtgc agcctgaagg ggtactcgaa ttcaacttcg atgttgaggg
     3721 cactctccgt cttgttggca cactccacgc tcagccgaag ctccccggtg tagctgccac
     3781 acgtagcaaa ggcgaagatg gcaaagaccc actgcagcac cttcacgaag ccaaggggct
     3841 ccttgaccac ccggaactga cccccagcca ccagctgatt caccacgtcc atgccaccgg
     3901 tggctctagc ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa
     3961 attatcactg ttattcttta gaatggtgca aagaataact tcgtataatg tatgctatac
     4021 gaagttatga attcgatatc aagcttatcg atgcccgaga taatcaacct ctggattaca
     4081 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     4141 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4201 ccttgtataa atcctggtta gttcttgcca cggcggaact catcgccgcc tgccttgccc
     4261 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttt atttgtgaaa
     4321 tttgtgatgc tattgcttta tttgtaacca tctagcttta tttgtgaaat ttgtgatgct
     4381 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4441 cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcgg ccgcaggaac
     4501 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     4561 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     4621 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4681 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     4741 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     4801 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4861 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4921 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4981 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5041 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     5101 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     5161 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5221 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5281 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5341 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5401 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5461 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5521 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5581 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5641 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5701 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5761 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5821 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5881 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5941 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6001 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6061 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6121 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6181 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6241 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     6301 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6361 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6421 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6481 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6541 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6601 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6661 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6721 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6781 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6841 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6901 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6961 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7021 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7081 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7141 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7201 cctggccttt tgctggcctt ttgctcacat gt
//
