LOCUS       sequence-212240-        6895 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF hCAR (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 6895)
  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 6895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 {hCAR}off-{ChETA-mRuby2}on-W3SL
FEATURES             Location/Qualifiers
     source          1..6895
                     /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             1884..1913
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1928..2638)
                     /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      2635..2644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2654..3580)
                     /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      3577..3586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3591..3624
                     /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    3675..3708
                     /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(3778..3798)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4158..4298
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4158..4287
                     /label=AAV2 ITR
     rep_origin      4373..4828
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4460..4479)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4670..4691
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4845..4864)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4964..4986
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5024..5042)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5110..5214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5215..5283,5284..6075)
                     /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(5433..5452)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6246..6834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6735..6754
                     /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 ggcgctcctg ctgtgcttcg tgctcctgtg cggagtagtg gatttcgcca
      841 gaagtttgag tatcactact cctgaagaga tgattgaaaa agccaaaggg gaaactgcct
      901 atctgccgtg caaatttacg cttagtcccg aagaccaggg accgctggac atcgagtggc
      961 tgatatcacc agctgataat cagaaggtgg atcaagtgat tattttatat tctggagaca
     1021 aaatttatga tgactactat ccagatctga aaggccgagt acattttacg agtaatgatc
     1081 tcaaatctgg tgatgcatca ataaatgtaa cgaatttaca actgtcagat attggcacat
     1141 atcagtgcaa agtgaaaaaa gctcctggtg ttgcaaataa gaagattcat ctggtagttc
     1201 ttgttaagcc ttcaggtgcg agatgttacg ttgatggatc tgaagaaatt ggaagtgact
     1261 ttaagataaa atgtgaacca aaagaaggtt cacttccatt acagtatgag tggcaaaaat
     1321 tgtctgactc acagaaaatg cccacttcat ggttagcaga aatgacttca tctgttatat
     1381 ctgtaaaaaa tgcctcttct gagtactctg ggacatacag ctgtacagtc agaaacagag
     1441 tgggctctga tcagtgcctg ttgcgtctaa acgttgtccc tccttcaaat aaagctggac
     1501 taattgcagg agccattata ggaactttgc ttgctctagc gctcattggt cttatcatct
     1561 tttgctgtcg taaaaagcgc agagaagaaa aatatgaaaa ggaagttcat cacgatatca
     1621 gggaagatgt gccacctcca aagagccgta cgtccactgc cagaagctac atcggcagta
     1681 atcattcatc cctggggtcc atgtctcctt ccaacatgga aggatattcc aagactcagt
     1741 ataaccaagt accaagtgaa gactttgaac gcactcctca gagtccgact ctcccacctg
     1801 ctaaggtagc tgcccctaat ctaagtcgaa tgggtgcgat tcctgtgatg attccagcac
     1861 agagcaagga tgggtctata gtagagcaga agctgatcag cgaggaggac ctgtaattaa
     1921 ttaattactt gtacagctcg tccatcccac caccaagccc ggcgaacttg gcaactgcgt
     1981 gttcgcgttg tactacgaac atttcattgt cactttcctc taacctttcc aggcggtgat
     2041 caacggcatg gataccgggc atcttgatgt tcccgacggt cttttttgac ctgtaagttg
     2101 ttacgaaaga gcaagacaga tggccaccac catcaacttt cagtgccata tgagtgtatc
     2161 ccctcagacc accatctgct ggatacatca tctctgtatt aggctcccaa cccttggtct
     2221 tcttctgcat cacgggacca ttggagggaa agtttacccc tctgacttgg acgtggtaaa
     2281 cgagacagcc atcctcaagg ctggtgtcct gcatgacggt gacgactcca ccatcttcgt
     2341 atctcgtaac tctttcccaa gtaaaaccct caggaaagga ctgtttaaag aaatcaggaa
     2401 tgcctttcgg gtacttgata aaagtacggc tgccatacat gaacgacgtg gcaagaatgt
     2461 caaaggcaaa tggcaggggt cctccctcga tgactttgat cctcatggtt tgagttccca
     2521 tgtacggatt gccttctcct tcacctgtgc atttgaattg gtggccgttg accgaacctt
     2581 ccatgaccac cttcatacgc atattttcct tgatcagctc ttcgccctta gacaccatgg
     2641 tggcggccgc tggcacggct ccggcctcgg cttcgtcttc gacgagagtc tcgacctcga
     2701 tctccgttcc gccgatgttc agtttggtgg ttttgcggat atctccgtga atcaatatgt
     2761 gctcgtggat caggacgcgc aggtagtgtc ccaacaaccc ccaacaattt ttactcatca
     2821 gatcaataat cgtgtgacct acggtggagc catagacgct caggacgcca aaaccttcgg
     2881 gccccaaaat gaagagaatt gggaacatac cccagctcac gaaaaacagc catgccatgc
     2941 cggtcacgac ctggcggcac cgaccctttg gcacagtatg ataaccctcg atatatgctt
     3001 tggcggcgtg aaaaaatgtg ttcgcgccat agcacaatcc aagacaaaag aagatgactt
     3061 taacatagcc ggttgccatg gcgctggtag ccccccacac gatagtcccg atgtctgaga
     3121 caaggagtcc catggttctc ctgctgtagt cgttgctcag gccggtgagg ttgctcaggc
     3181 ggataaggat gacaggacaa gtgagcagcc aggttgcata gcgcagccac tgcacccggt
     3241 gtcctgtggc aaggtagagc atagagggat tcttaaactc aaaaaagaac tcgagaatca
     3301 ccttaaccat ttcaatggcg cacacataga tctcctccca gccgcatgta gatttccagg
     3361 tttggtaggc atagaacatc agcagcaaaa tgctgaatcc tgctgcaagc cactgcagga
     3421 catttgacgc ggtctgagcg ccgttcgtgc cgcgagattc aatccatccg gcacagtaac
     3481 attgatcctc agggaccagg acggacccgt tcaccaccac aggattagta acgaacaaaa
     3541 gttcgcgtcc gacggcagac aaagcgccgc catagtccat ggtggctagc ataacttcgt
     3601 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     3661 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attccgctcg
     3721 agataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3781 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3841 ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga
     3901 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3961 ttccgtggtg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct
     4021 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4081 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     4141 ttttttaaag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4201 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4261 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4321 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4381 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4441 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4501 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4561 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4621 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4681 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4741 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4801 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4861 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4921 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4981 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5041 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5101 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5161 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5221 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5281 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5341 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5401 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5461 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5521 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5581 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5641 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5701 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5761 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5821 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5881 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5941 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6001 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6061 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6121 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6361 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
