LOCUS       sequence-209261-        7286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF mCAR (for efficient 
            CAV-2 infection) and Cre-ON GCaMP6f (ultrasensitive calcium sensor).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7286)
  AUTHORS   Li SJ, Vaughan A, Sturgill JF, Kepecs A
  TITLE     A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism 
            for Efficient Retrograde Targeting of Neurons.
  JOURNAL   Neuron. 2018 Jun 6;98(5):905-917.e5. doi: 
            10.1016/j.neuron.2018.05.028.
  PUBMED    29879392
REFERENCE   2  (bases 1 to 7286)
  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"; doi: 
            "10.1016/j.neuron.2018.05"; journalName: "Neuron"; date: "2018-06-6-
            6"; volume: "98"; issue: "5"; pages: "905-917.e5"
COMMENT     Sequence Label: pAAV-hSyn-DIO {mCAR}off-{GCaMP6f}on-WPRE
FEATURES             Location/Qualifiers
     source          1..7286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..142
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   13..142
                     /label=AAV2 ITR
     promoter        187..634
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(672..705)
                     /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(756..789)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      795..804
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1857..1886
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(1898..2341,2342..2347,2348..2782,2783..2800,
                     2801..3070,3071..3076,3077..3133,3134..3160,3161..3184,
                     3185..3187,3188..3220,3221..3223,3224..3241,3242..3250))
                     /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"
     primer_bind     2457..2476
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2716..2737
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2843..2864)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3145..3154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3247..3256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3272..3305
                     /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    3356..3389
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3414..4002
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3467..3487)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3885..3896)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4034..4510
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4233..4252)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4550..4690
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4550..4679
                     /label=AAV2 ITR
     rep_origin      4765..5220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4852..4871)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5062..5083
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5237..5256)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5356..5378
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5416..5434)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5502..5606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5607..5675,5676..6467)
                     /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(5825..5844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6638..7226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7127..7146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
       61 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      121 aactccatca ctaggggttc ctgcggccgc acgcgtgtgt ctagactgca gagggccctg
      181 cgtatgagtg caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac
      241 gaccgacccc gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct
      301 atcagagagg gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc
      361 accgcggaca gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga
      421 aggcgcgctg acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg
      481 cgtccgcgcc gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca
      541 cgggcgcgac catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg
      601 cagcggagga gtcgtgtcgt gcctgagagc gcagtcgaga aggtaccgga tcctctagag
      661 tcgactccgg aataacttcg tataggatac tttatacgaa gttatgcaga atggtagctg
      721 gattgtagct gctattagca atatgaaacc tcttaataac ttcgtatagc atacattata
      781 cgaagttatg gcgcgccacc atggctcgcc tactgtgctt cgtgctcttg tgcgggatcg
      841 cggatttcac cagtggtttg agcatcacta cacccgaaca gaggatcgaa aaagccaaag
      901 gggaaactgc gtatctacca tgcaagttta ctctcagtcc cgaagaccag ggaccactgg
      961 acattgaatg gctgatatcc ccgtctgata accagatagt ggatcaagtg atcattttgt
     1021 attctggaga caaaatttat gataactact atccggatct gaaaggacgg gtacatttta
     1081 cgagtaacga tgtcaagtct ggcgacgcat ctataaatgt gaccaacctg cagctgtcgg
     1141 acattggcac ttaccagtgc aaagtgaaga aagcccctgg ggttgcaaat aagaaattcc
     1201 tgctgaccgt tcttgttaag ccttcaggta caagatgctt cgtggatgga tcggaagaga
     1261 ttggaaatga cttcaagcta aaatgtgaac ccaaggaagg ctcccttcca ctacagtttg
     1321 aatggcagaa actgtcggac tcccagacaa tgcctacgcc atggctggca gaaatgacgt
     1381 caccagttat atctgtgaag aacgccagtt ctgagtattc tgggacatac agctgcacgg
     1441 ttcaaaacag agtgggctct gaccagtgta tgctgcgact agacgttgtc ccaccctcca
     1501 accgagccgg aacgatcgcg ggcgccgtca tagggacgct gctggccctt gtgctcatcg
     1561 gggccatcct cttctgctgt cacaggaaac gcagagaaga gaagtacgag aaggaagttc
     1621 atcatgatat cagggaagat gtgcctcctc caaagagtcg gacatccact gccaggagct
     1681 atattggcag caaccattcc tccctgggat ccatgtcccc ctctaacatg gaggggtatt
     1741 ccaagacgca gtataaccaa gtccccagtg aagactttga acgtgcgcct cagagcccga
     1801 ctctggcacc cgctaagttc aagtacgctt acaagaccga tggcattaca gtggtagagc
     1861 agaagctgat cagcgaggag gacctgtaat taattaatta cttcgctgtc atcatttgta
     1921 caaactcttc gtagtttacc tgaccatccc catcgatgtc tgcttccctg atcatttcat
     1981 caacctcttc atctgttaac ttctctccaa ggtttgtcat cacgtggcga agctctgctg
     2041 cactgatgta gccattgcca tccttatcaa acacaccgaa cgcttctcta atttcttctt
     2101 ccgtgtccct gtatttcatt tttcttgcca tcattgtcag gaactcaggg aagtcgattg
     2161 tgccgtcacc gtcggcatct acttcattga tcatgtcctg cagctctgct tctgtggggt
     2221 tctgccccag agaccgcatc accgtcccca gctccttggt tgttattgtc ccatccccgt
     2281 ccttgtcaaa tagggagaat tcctctttaa attctgcgat ctgctcttca gtcagttggt
     2341 ccggcaggtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
     2401 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
     2461 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gatgtagcct tcgggcatgg
     2521 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
     2581 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     2641 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc accctcgccg gacacgctga
     2701 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     2761 gctcctcgcc cttgctcacc atgctccctc cggtaccgcc cttgtacagc tcgtccatgc
     2821 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     2881 tggggtcttt cgaaagtttg gactgcacgc tcaggtagtg gttgtcgggc agcagcacgg
     2941 ggccgtcgcc gatgggggtg ttctgctggt agtggtaggc gagctgcacg ccgccgtcct
     3001 cgatgttgtg gcggatcttg aagttcgcct tgatgccgtt cttctgcttg tcggccttga
     3061 tatagacgtt ctcgagtgag ctcagccgac ctatagctct gactgcgtga cctgtcttat
     3121 tccacttacg acgtgatgag tcgaccatgg tggcgagatc cttatcgtca tcgtcgtaca
     3181 gatcccgacc catttgctgt ccaccagtca tgctagccat accatgatga tgatgatgat
     3241 gagaacccat ggtggcaaca gcgctactag cataacttcg tataaagtat cctatacgaa
     3301 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     3361 ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac
     3421 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3481 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3541 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3601 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3661 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3721 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3781 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg
     3841 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3901 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     3961 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc
     4021 tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga
     4081 agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc
     4141 tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca
     4201 agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt
     4261 ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca
     4321 gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt
     4381 ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt
     4441 gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt
     4501 ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta
     4561 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4621 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4681 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4741 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4801 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4861 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4921 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4981 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5041 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5101 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5161 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5221 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5281 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5341 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5401 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5461 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5521 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5581 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5641 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5701 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5761 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5821 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5881 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5941 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6001 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6061 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6781 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7261 ccttttgctg gccttttgct cacatg
//
