LOCUS       Exported                6913 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF hCAR (for efficient 
            CAV-2 infection), Cre-ON ChETA-EYFP (for optogenetic activation), 
            and W3SL regulatory cassette (for maximize cloning capacity).
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO {hCAR}off-{ChETA-EYFP}on-W3SL
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6913)
  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 6913)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6913
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    671..704
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    755..788
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      794..803
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1895..1924
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1936..2655)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1981..2002)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2330..2349
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2589..2610
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2652..2661
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(2671..3597)
                     /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      3594..3603
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3608..3641)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(3692..3725)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(3795..3815)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4175..4315
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4175..4304
                     /label=AAV2 ITR
     rep_origin      4390..4845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4477..4496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4687..4708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4862..4881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4981..5003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5041..5059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5127..5231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5232..6092
                     /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(5450..5469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6752..6771
                     /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 tggcgctcct gctgtgcttc gtgctcctgt gcggagtagt
      841 ggatttcgcc agaagtttga gtatcactac tcctgaagag atgattgaaa aagccaaagg
      901 ggaaactgcc tatctgccgt gcaaatttac gcttagtccc gaagaccagg gaccgctgga
      961 catcgagtgg ctgatatcac cagctgataa tcagaaggtg gatcaagtga ttattttata
     1021 ttctggagac aaaatttatg atgactacta tccagatctg aaaggccgag tacattttac
     1081 gagtaatgat ctcaaatctg gtgatgcatc aataaatgta acgaatttac aactgtcaga
     1141 tattggcaca tatcagtgca aagtgaaaaa agctcctggt gttgcaaata agaagattca
     1201 tctggtagtt cttgttaagc cttcaggtgc gagatgttac gttgatggat ctgaagaaat
     1261 tggaagtgac tttaagataa aatgtgaacc aaaagaaggt tcacttccat tacagtatga
     1321 gtggcaaaaa ttgtctgact cacagaaaat gcccacttca tggttagcag aaatgacttc
     1381 atctgttata tctgtaaaaa atgcctcttc tgagtactct gggacataca gctgtacagt
     1441 cagaaacaga gtgggctctg atcagtgcct gttgcgtcta aacgttgtcc ctccttcaaa
     1501 taaagctgga ctaattgcag gagccattat aggaactttg cttgctctag cgctcattgg
     1561 tcttatcatc ttttgctgtc gtaaaaagcg cagagaagaa aaatatgaaa aggaagttca
     1621 tcacgatatc agggaagatg tgccacctcc aaagagccgt acgtccactg ccagaagcta
     1681 catcggcagt aatcattcat ccctggggtc catgtctcct tccaacatgg aaggatattc
     1741 caagactcag tataaccaag taccaagtga agactttgaa cgcactcctc agagtccgac
     1801 tctcccacct gctaaggtag ctgcccctaa tctaagtcga atgggtgcga ttcctgtgat
     1861 gattccagca cagagcaagg atgggtctat agtagagcag aagctgatca gcgaggagga
     1921 cctgtaatta attaattact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     1981 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     2041 ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     2101 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     2161 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     2221 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     2281 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     2341 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     2401 gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc
     2461 gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     2521 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     2581 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2641 gcccttgctc accatggtgg cggccgctgg cacggctccg gcctcggctt cgtcttcgac
     2701 gagagtctcg acctcgatct ccgttccgcc gatgttcagt ttggtggttt tgcggatatc
     2761 tccgtgaatc aatatgtgct cgtggatcag gacgcgcagg tagtgtccca acaaccccca
     2821 acaattttta ctcatcagat caataatcgt gtgacctacg gtggagccat agacgctcag
     2881 gacgccaaaa ccttcgggcc ccaaaatgaa gagaattggg aacatacccc agctcacgaa
     2941 aaacagccat gccatgccgg tcacgacctg gcggcaccga ccctttggca cagtatgata
     3001 accctcgata tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag
     3061 acaaaagaag atgactttaa catagccggt tgccatggcg ctggtagccc cccacacgat
     3121 agtcccgatg tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc
     3181 ggtgaggttg ctcaggcgga taaggatgac aggacaagtg agcagccagg ttgcatagcg
     3241 cagccactgc acccggtgtc ctgtggcaag gtagagcata gagggattct taaactcaaa
     3301 aaagaactcg agaatcacct taaccatttc aatggcgcac acatagatct cctcccagcc
     3361 gcatgtagat ttccaggttt ggtaggcata gaacatcagc agcaaaatgc tgaatcctgc
     3421 tgcaagccac tgcaggacat ttgacgcggt ctgagcgccg ttcgtgccgc gagattcaat
     3481 ccatccggca cagtaacatt gatcctcagg gaccaggacg gacccgttca ccaccacagg
     3541 attagtaacg aacaaaagtt cgcgtccgac ggcagacaaa gcgccgccat agtccatggt
     3601 ggctagcata acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt
     3661 atcactgtta ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa
     3721 gttatgaatt ccgctcgaga taatcaacct ctggattaca aaatttgtga aagattgact
     3781 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3841 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta
     3901 gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3961 ctgttgggca ctgacaattc cgtggtgttt atttgtgaaa tttgtgatgc tattgcttta
     4021 tttgtaacca tctagcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     4081 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     4141 gggggagatg tgggaggttt tttaaagcgg ccgcaggaac ccctagtgat ggagttggcc
     4201 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4261 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4321 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4381 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4441 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4501 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4561 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4621 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4681 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     4741 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     4801 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4861 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4921 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4981 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5041 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5461 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5941 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6901 ttgctcacat gtt
//
