LOCUS       sequence-212377-        6856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF mCAR (for efficient 
            CAV-2 infection), Cre-ON ChETA-mRuby2 (for optogenetic activation), 
            and W3SL regulatory cassette (for maximize cloning capacity).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6856)
  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 6856)
  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-{ChETA-mRuby2}on-W3SL
FEATURES             Location/Qualifiers
     source          1..6856
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /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(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      783..792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1845..1874
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1889..2599)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     regulatory      2596..2605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2615..3541)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYATWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3538..3547
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3552..3585
                     /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    3636..3669
                     /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(3739..3759)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4119..4259
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4119..4248
                     /label=AAV2 ITR
     rep_origin      4334..4789
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4421..4440)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4631..4652
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4806..4825)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4925..4947
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4985..5003)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5071..5175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5176..5244,5245..6036)
                     /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(5394..5413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6207..6795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6696..6715
                     /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 gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccaccat ggctcgccta ctgtgcttcg tgctcttgtg cgggatcgcg gatttcacca
      841 gtggtttgag catcactaca cccgaacaga ggatcgaaaa agccaaaggg gaaactgcgt
      901 atctaccatg caagtttact ctcagtcccg aagaccaggg accactggac attgaatggc
      961 tgatatcccc gtctgataac cagatagtgg atcaagtgat cattttgtat tctggagaca
     1021 aaatttatga taactactat ccggatctga aaggacgggt acattttacg agtaacgatg
     1081 tcaagtctgg cgacgcatct ataaatgtga ccaacctgca gctgtcggac attggcactt
     1141 accagtgcaa agtgaagaaa gcccctgggg ttgcaaataa gaaattcctg ctgaccgttc
     1201 ttgttaagcc ttcaggtaca agatgcttcg tggatggatc ggaagagatt ggaaatgact
     1261 tcaagctaaa atgtgaaccc aaggaaggct cccttccact acagtttgaa tggcagaaac
     1321 tgtcggactc ccagacaatg cctacgccat ggctggcaga aatgacgtca ccagttatat
     1381 ctgtgaagaa cgccagttct gagtattctg ggacatacag ctgcacggtt caaaacagag
     1441 tgggctctga ccagtgtatg ctgcgactag acgttgtccc accctccaac cgagccggaa
     1501 cgatcgcggg cgccgtcata gggacgctgc tggcccttgt gctcatcggg gccatcctct
     1561 tctgctgtca caggaaacgc agagaagaga agtacgagaa ggaagttcat catgatatca
     1621 gggaagatgt gcctcctcca aagagtcgga catccactgc caggagctat attggcagca
     1681 accattcctc cctgggatcc atgtccccct ctaacatgga ggggtattcc aagacgcagt
     1741 ataaccaagt ccccagtgaa gactttgaac gtgcgcctca gagcccgact ctggcacccg
     1801 ctaagttcaa gtacgcttac aagaccgatg gcattacagt ggtagagcag aagctgatca
     1861 gcgaggagga cctgtaatta attaattact tgtacagctc gtccatccca ccaccaagcc
     1921 cggcgaactt ggcaactgcg tgttcgcgtt gtactacgaa catttcattg tcactttcct
     1981 ctaacctttc caggcggtga tcaacggcat ggataccggg catcttgatg ttcccgacgg
     2041 tcttttttga cctgtaagtt gttacgaaag agcaagacag atggccacca ccatcaactt
     2101 tcagtgccat atgagtgtat cccctcagac caccatctgc tggatacatc atctctgtat
     2161 taggctccca acccttggtc ttcttctgca tcacgggacc attggaggga aagtttaccc
     2221 ctctgacttg gacgtggtaa acgagacagc catcctcaag gctggtgtcc tgcatgacgg
     2281 tgacgactcc accatcttcg tatctcgtaa ctctttccca agtaaaaccc tcaggaaagg
     2341 actgtttaaa gaaatcagga atgcctttcg ggtacttgat aaaagtacgg ctgccataca
     2401 tgaacgacgt ggcaagaatg tcaaaggcaa atggcagggg tcctccctcg atgactttga
     2461 tcctcatggt ttgagttccc atgtacggat tgccttctcc ttcacctgtg catttgaatt
     2521 ggtggccgtt gaccgaacct tccatgacca ccttcatacg catattttcc ttgatcagct
     2581 cttcgccctt agacaccatg gtggcggccg ctggcacggc tccggcctcg gcttcgtctt
     2641 cgacgagagt ctcgacctcg atctccgttc cgccgatgtt cagtttggtg gttttgcgga
     2701 tatctccgtg aatcaatatg tgctcgtgga tcaggacgcg caggtagtgt cccaacaacc
     2761 cccaacaatt tttactcatc agatcaataa tcgtgtgacc tacggtggag ccatagacgc
     2821 tcaggacgcc aaaaccttcg ggccccaaaa tgaagagaat tgggaacata ccccagctca
     2881 cgaaaaacag ccatgccatg ccggtcacga cctggcggca ccgacccttt ggcacagtat
     2941 gataaccctc gatatatgct ttggcggcgt gaaaaaatgt gttcgcgcca tagcacaatc
     3001 caagacaaaa gaagatgact ttaacatagc cggttgccat ggcgctggta gccccccaca
     3061 cgatagtccc gatgtctgag acaaggagtc ccatggttct cctgctgtag tcgttgctca
     3121 ggccggtgag gttgctcagg cggataagga tgacaggaca agtgagcagc caggttgcat
     3181 agcgcagcca ctgcacccgg tgtcctgtgg caaggtagag catagaggga ttcttaaact
     3241 caaaaaagaa ctcgagaatc accttaacca tttcaatggc gcacacatag atctcctccc
     3301 agccgcatgt agatttccag gtttggtagg catagaacat cagcagcaaa atgctgaatc
     3361 ctgctgcaag ccactgcagg acatttgacg cggtctgagc gccgttcgtg ccgcgagatt
     3421 caatccatcc ggcacagtaa cattgatcct cagggaccag gacggacccg ttcaccacca
     3481 caggattagt aacgaacaaa agttcgcgtc cgacggcaga caaagcgccg ccatagtcca
     3541 tggtggctag cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga
     3601 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata
     3661 cgaagttatg aattccgctc gagataatca acctctggat tacaaaattt gtgaaagatt
     3721 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3781 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3841 gttagttctt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3901 tcggctgttg ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc
     3961 tttatttgta accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     4021 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     4081 ttcaggggga gatgtgggag gttttttaaa gcggccgcag gaacccctag tgatggagtt
     4141 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     4201 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     4261 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     4321 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4381 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     4441 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4501 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     4561 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4621 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     4681 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     4741 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4801 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4861 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4921 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4981 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5041 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5101 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5161 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5221 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5281 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5341 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5401 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5461 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5521 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5581 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5641 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5701 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5761 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5821 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5881 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5941 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6001 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6061 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6121 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6181 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6241 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6301 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6361 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6421 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6481 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6541 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6601 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6661 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6721 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6781 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6841 ccttttgctc acatgt
//
