LOCUS       sequence-209254-        6868 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 6868)
  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 6868)
  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..6868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        186..633
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(671..704)
                     /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(755..788)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      794..803
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1856..1885
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1900..2610)
                     /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      2607..2616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2626..3552)
                     /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      3549..3558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3563..3596
                     /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    3647..3680
                     /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(3750..3770)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4130..4270
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4130..4259
                     /label=AAV2 ITR
     rep_origin      4345..4800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4432..4451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4642..4663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4817..4836)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4936..4958
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4996..5014)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5082..5186
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5187..5255,5256..6047)
                     /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(5405..5424)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6218..6806
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6707..6726
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagactgcag agggccctgc
      181 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      241 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      301 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      361 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
      421 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
      481 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
      541 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
      601 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt
      661 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
      721 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
      781 gaagttatgg cgcgccacca tggctcgcct actgtgcttc gtgctcttgt gcgggatcgc
      841 ggatttcacc agtggtttga gcatcactac acccgaacag aggatcgaaa aagccaaagg
      901 ggaaactgcg tatctaccat gcaagtttac tctcagtccc gaagaccagg gaccactgga
      961 cattgaatgg ctgatatccc cgtctgataa ccagatagtg gatcaagtga tcattttgta
     1021 ttctggagac aaaatttatg ataactacta tccggatctg aaaggacggg tacattttac
     1081 gagtaacgat gtcaagtctg gcgacgcatc tataaatgtg accaacctgc agctgtcgga
     1141 cattggcact taccagtgca aagtgaagaa agcccctggg gttgcaaata agaaattcct
     1201 gctgaccgtt cttgttaagc cttcaggtac aagatgcttc gtggatggat cggaagagat
     1261 tggaaatgac ttcaagctaa aatgtgaacc caaggaaggc tcccttccac tacagtttga
     1321 atggcagaaa ctgtcggact cccagacaat gcctacgcca tggctggcag aaatgacgtc
     1381 accagttata tctgtgaaga acgccagttc tgagtattct gggacataca gctgcacggt
     1441 tcaaaacaga gtgggctctg accagtgtat gctgcgacta gacgttgtcc caccctccaa
     1501 ccgagccgga acgatcgcgg gcgccgtcat agggacgctg ctggcccttg tgctcatcgg
     1561 ggccatcctc ttctgctgtc acaggaaacg cagagaagag aagtacgaga aggaagttca
     1621 tcatgatatc agggaagatg tgcctcctcc aaagagtcgg acatccactg ccaggagcta
     1681 tattggcagc aaccattcct ccctgggatc catgtccccc tctaacatgg aggggtattc
     1741 caagacgcag tataaccaag tccccagtga agactttgaa cgtgcgcctc agagcccgac
     1801 tctggcaccc gctaagttca agtacgctta caagaccgat ggcattacag tggtagagca
     1861 gaagctgatc agcgaggagg acctgtaatt aattaattac ttgtacagct cgtccatccc
     1921 accaccaagc ccggcgaact tggcaactgc gtgttcgcgt tgtactacga acatttcatt
     1981 gtcactttcc tctaaccttt ccaggcggtg atcaacggca tggataccgg gcatcttgat
     2041 gttcccgacg gtcttttttg acctgtaagt tgttacgaaa gagcaagaca gatggccacc
     2101 accatcaact ttcagtgcca tatgagtgta tcccctcaga ccaccatctg ctggatacat
     2161 catctctgta ttaggctccc aacccttggt cttcttctgc atcacgggac cattggaggg
     2221 aaagtttacc cctctgactt ggacgtggta aacgagacag ccatcctcaa ggctggtgtc
     2281 ctgcatgacg gtgacgactc caccatcttc gtatctcgta actctttccc aagtaaaacc
     2341 ctcaggaaag gactgtttaa agaaatcagg aatgcctttc gggtacttga taaaagtacg
     2401 gctgccatac atgaacgacg tggcaagaat gtcaaaggca aatggcaggg gtcctccctc
     2461 gatgactttg atcctcatgg tttgagttcc catgtacgga ttgccttctc cttcacctgt
     2521 gcatttgaat tggtggccgt tgaccgaacc ttccatgacc accttcatac gcatattttc
     2581 cttgatcagc tcttcgccct tagacaccat ggtggcggcc gctggcacgg ctccggcctc
     2641 ggcttcgtct tcgacgagag tctcgacctc gatctccgtt ccgccgatgt tcagtttggt
     2701 ggttttgcgg atatctccgt gaatcaatat gtgctcgtgg atcaggacgc gcaggtagtg
     2761 tcccaacaac ccccaacaat ttttactcat cagatcaata atcgtgtgac ctacggtgga
     2821 gccatagacg ctcaggacgc caaaaccttc gggccccaaa atgaagagaa ttgggaacat
     2881 accccagctc acgaaaaaca gccatgccat gccggtcacg acctggcggc accgaccctt
     2941 tggcacagta tgataaccct cgatatatgc tttggcggcg tgaaaaaatg tgttcgcgcc
     3001 atagcacaat ccaagacaaa agaagatgac tttaacatag ccggttgcca tggcgctggt
     3061 agccccccac acgatagtcc cgatgtctga gacaaggagt cccatggttc tcctgctgta
     3121 gtcgttgctc aggccggtga ggttgctcag gcggataagg atgacaggac aagtgagcag
     3181 ccaggttgca tagcgcagcc actgcacccg gtgtcctgtg gcaaggtaga gcatagaggg
     3241 attcttaaac tcaaaaaaga actcgagaat caccttaacc atttcaatgg cgcacacata
     3301 gatctcctcc cagccgcatg tagatttcca ggtttggtag gcatagaaca tcagcagcaa
     3361 aatgctgaat cctgctgcaa gccactgcag gacatttgac gcggtctgag cgccgttcgt
     3421 gccgcgagat tcaatccatc cggcacagta acattgatcc tcagggacca ggacggaccc
     3481 gttcaccacc acaggattag taacgaacaa aagttcgcgt ccgacggcag acaaagcgcc
     3541 gccatagtcc atggtggcta gcataacttc gtataaagta tcctatacga agttatttgc
     3601 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     3661 tgtatgctat acgaagttat gaattccgct cgagataatc aacctctgga ttacaaaatt
     3721 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3781 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3841 tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3901 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgtttatttg tgaaatttgt
     3961 gatgctattg ctttatttgt aaccatctag ctttatttgt gaaatttgtg atgctattgc
     4021 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     4081 tatgtttcag gttcaggggg agatgtggga ggttttttaa agcggccgca ggaaccccta
     4141 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4201 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4261 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4321 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4381 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4441 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4501 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4561 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4621 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     4681 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     4741 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     4801 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     4861 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4921 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4981 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5041 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5101 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5161 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5221 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5281 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5341 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5401 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5461 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5521 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5581 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5641 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5701 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5761 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5821 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5881 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5941 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6001 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6061 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6121 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6181 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6241 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6301 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6361 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6421 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6481 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6541 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6601 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6661 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6721 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6781 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6841 ccttttgctg gccttttgct cacatgtt
//
