LOCUS       Exported                7410 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF hCAR (for efficient 
            CAV-2 infection), Cre-ON hM4Di-mCherry (Gi-coupled DREADD for 
            neuronal silencing), W3SL cassette (for maximize cloning capacity).
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO {hCAR}off-{hM4Di-mCherry}on-W3SL
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7410)
  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 7410)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7410
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   62..191
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        236..683
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    721..754
                     /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    805..838
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      844..853
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1945..1974
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1986..2696)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2264..2283)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2533..2551
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2693..2702
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      4151..4160
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4167..4200)
                     /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(4251..4284)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4354..4374)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4734..4874
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4734..4863
                     /label=AAV2 ITR
     rep_origin      4949..5404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5246..5267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5421..5440)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5540..5562
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5600..5618)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5686..5790
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5791..6651
                     /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(6009..6028)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6822..7410
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7311..7330
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
      121 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      181 taggggttcc tgcggccgca cgcgtgtgtc tagactgcag agggccctgc gtatgagtgc
      241 aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg
      301 acccactgga caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg
      361 ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag
      421 tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga
      481 cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg
      541 ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc
      601 atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag
      661 tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt cgactccgga
      721 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
      781 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
      841 cgcgccacca tggcgctcct gctgtgcttc gtgctcctgt gcggagtagt ggatttcgcc
      901 agaagtttga gtatcactac tcctgaagag atgattgaaa aagccaaagg ggaaactgcc
      961 tatctgccgt gcaaatttac gcttagtccc gaagaccagg gaccgctgga catcgagtgg
     1021 ctgatatcac cagctgataa tcagaaggtg gatcaagtga ttattttata ttctggagac
     1081 aaaatttatg atgactacta tccagatctg aaaggccgag tacattttac gagtaatgat
     1141 ctcaaatctg gtgatgcatc aataaatgta acgaatttac aactgtcaga tattggcaca
     1201 tatcagtgca aagtgaaaaa agctcctggt gttgcaaata agaagattca tctggtagtt
     1261 cttgttaagc cttcaggtgc gagatgttac gttgatggat ctgaagaaat tggaagtgac
     1321 tttaagataa aatgtgaacc aaaagaaggt tcacttccat tacagtatga gtggcaaaaa
     1381 ttgtctgact cacagaaaat gcccacttca tggttagcag aaatgacttc atctgttata
     1441 tctgtaaaaa atgcctcttc tgagtactct gggacataca gctgtacagt cagaaacaga
     1501 gtgggctctg atcagtgcct gttgcgtcta aacgttgtcc ctccttcaaa taaagctgga
     1561 ctaattgcag gagccattat aggaactttg cttgctctag cgctcattgg tcttatcatc
     1621 ttttgctgtc gtaaaaagcg cagagaagaa aaatatgaaa aggaagttca tcacgatatc
     1681 agggaagatg tgccacctcc aaagagccgt acgtccactg ccagaagcta catcggcagt
     1741 aatcattcat ccctggggtc catgtctcct tccaacatgg aaggatattc caagactcag
     1801 tataaccaag taccaagtga agactttgaa cgcactcctc agagtccgac tctcccacct
     1861 gctaaggtag ctgcccctaa tctaagtcga atgggtgcga ttcctgtgat gattccagca
     1921 cagagcaagg atgggtctat agtagagcag aagctgatca gcgaggagga cctgtaatta
     1981 attaattact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
     2041 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
     2101 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
     2161 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     2221 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     2281 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     2341 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     2401 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     2461 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     2521 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     2581 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     2641 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
     2701 gcgaccggtg gatcccgcct ggcagtgccg atgttccgat actggcacag cagcaggtgc
     2761 cggaaggtct ttttaaaggt ggcgttgcac agagcatagc aggcagggtt gatggtgctg
     2821 ttgacgtagc agagccagta gccaatggac cacaccgtgt cagggatgca gctctggcag
     2881 aaggtgttca ccaggaccat gacgttgtag ggcgtccagg tgaggatgaa ggccagcaga
     2941 atggcaaaga tcgttcgtgt cactttgcgc tcccgggccg ccatctgccg cttcttgcgc
     3001 acctggttgc gagcgatgct ggcgaacttg cgggccacgt tggccgcagg gcgcatgcca
     3061 gccggcgtgg caggcacaat ctcaatggct gtcacacact cattgcctgt ctgcttcgtc
     3121 acaatctgga tcttggacca tctggaggct gggttgaggg cccgcggctg caggggaggg
     3181 gcgggcatgg cgggcgtggt ggcctctgtg gtggacagct ctgtggctgg gcgttccttg
     3241 gtgttctggg tggcactgcc tgagctggac tcattggaag tgtccttatc agccacgggg
     3301 cgcggtggcg gtggcagcgc tggcgggggg gcctcctcca gcttgccatt gcgcagctcc
     3361 tcccgggcgg cctccccggg cgggggcttc ttgacgctct gcttcattag tgggctcttg
     3421 aggaaggcca gcgtcttggc tttcttctcc ttcgggccct cgggccggtg cttgtggact
     3481 cggctgcgac tggccaggga gatgtggatg tacagcaccg tcatgatgac cacaggcagg
     3541 tagaagccag caatggctgt gccaaaggtc actgctgggt tggacaggaa ctggatgaag
     3601 cactggttgt cgggcaccgt ccgcttaccc accacaaact gccagaacaa gatggcaggc
     3661 gcccagagca cgaaggacag tacccaggca gcagcaatca tgaggcctgc catcttggtg
     3721 gtgcgccggg cagggtaggt gagaggcttg gtgacgcaga agtagcggtc aaagctgatg
     3781 atgagaaggt tcatgacgga ggcgttgctc accacgcagt ccagggccag ccacaggtcg
     3841 cagaccacgg cgcccagggg ccagtagccc ttgatgatgt acacggtgta gaggttcatg
     3901 gagaaggcgc ctatgatgag atcagcacac gccaggctga agaggaagta gttgttgact
     3961 gtctgcagct gcctgttgac cttgatggac agcatcacca ggatgttgcc cacgacagtc
     4021 accaggctca gggagcctgt cactgtggca atgaagacca tttccaccgt ctcatagcga
     4081 ttgtgggatg atgacgtgac caggcgcacg gactgattgc ccgagctgcc attgacaggt
     4141 gtgaagttgg ccatggtggc gctagcataa cttcgtataa agtatcctat acgaagttat
     4201 ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt
     4261 ataatgtatg ctatacgaag ttatgaattc cgctcgagat aatcaacctc tggattacaa
     4321 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     4381 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     4441 cttgtataaa tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg
     4501 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat
     4561 ttgtgatgct attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta
     4621 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4681 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcggc cgcaggaacc
     4741 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4801 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4861 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4921 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4981 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     5041 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5101 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5161 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5221 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5281 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5341 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5401 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5461 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5521 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5581 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5641 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5701 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5761 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5821 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5881 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5941 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6001 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6061 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6121 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6181 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6241 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6301 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6361 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6421 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6481 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6541 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6601 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6661 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6721 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6781 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6841 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6901 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6961 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7021 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7081 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7141 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7201 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7261 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7321 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7381 tgctcgtcag gggggcggag cctatggaaa
//
