LOCUS       Exported                6615 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector with hSynapsin promoter, Cre-OFF hCAR (for efficient 
            CAV-2 infection), Cre-ON GCaMP6f (ultrasensitive calcium sensor), 
            and W3SL regulatory cassette (for maximize cloning capacity).
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO {hCAR}off-{GCaMP6f}on-W3SL
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6615)
  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 6615)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6615
                     /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..3288)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2495..2514
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2754..2775
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2881..2902)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3183..3192
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     regulatory      3285..3294
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3310..3343)
                     /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(3394..3427)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(3497..3517)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3877..4017
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3877..4006
                     /label=AAV2 ITR
     rep_origin      4092..4547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4179..4198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4389..4410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4564..4583)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4683..4705
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4743..4761)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4829..4933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4934..5794
                     /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(5152..5171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5965..6553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6454..6473
                     /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 tcgctgtcat catttgtaca aactcttcgt agtttacctg
     1981 accatcccca tcgatgtctg cttccctgat catttcatca acctcttcat ctgttaactt
     2041 ctctccaagg tttgtcatca cgtggcgaag ctctgctgca ctgatgtagc cattgccatc
     2101 cttatcaaac acaccgaacg cttctctaat ttcttcttcc gtgtccctgt atttcatttt
     2161 tcttgccatc attgtcagga actcagggaa gtcgattgtg ccgtcaccgt cggcatctac
     2221 ttcattgatc atgtcctgca gctctgcttc tgtggggttc tgccccagag accgcatcac
     2281 cgtccccagc tccttggttg ttattgtccc atccccgtcc ttgtcaaata gggagaattc
     2341 ctctttaaat tctgcgatct gctcttcagt cagttggtcc ggcaggttgt actccagctt
     2401 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac
     2461 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa
     2521 gaagatggtg cgctcctgga tgtagccttc gggcatggcg gacttgaaga agtcgtgctg
     2581 cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag
     2641 ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc
     2701 gtaggtggca tcgccctcac cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc
     2761 gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat
     2821 gctccctccg gtaccgccct tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     2881 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttcg aaagtttgga
     2941 ctgcacgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     3001 ctgctggtag tggtaggcga gctgcacgcc gccgtcctcg atgttgtggc ggatcttgaa
     3061 gttcgccttg atgccgttct tctgcttgtc ggccttgata tagacgttct cgagtgagct
     3121 cagccgacct atagctctga ctgcgtgacc tgtcttattc cacttacgac gtgatgagtc
     3181 gaccatggtg gcgagatcct tatcgtcatc gtcgtacaga tcccgaccca tttgctgtcc
     3241 accagtcatg ctagccatac catgatgatg atgatgatga gaacccatgg tggcaacagc
     3301 gctactagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     3361 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     3421 aagttatgaa ttccgctcga gataatcaac ctctggatta caaaatttgt gaaagattga
     3481 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3541 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3601 tagttcttgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3661 ggctgttggg cactgacaat tccgtggtgt ttatttgtga aatttgtgat gctattgctt
     3721 tatttgtaac catctagctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3781 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3841 cagggggaga tgtgggaggt tttttaaagc ggccgcagga acccctagtg atggagttgg
     3901 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     3961 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     4021 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     4081 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4141 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4201 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4261 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     4321 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4381 ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc
     4441 ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta
     4501 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     4561 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4621 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4681 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4741 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     4801 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4861 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     4921 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     4981 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5041 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5101 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5161 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5221 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5281 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5341 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5401 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5461 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5521 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5581 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5641 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5701 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     5761 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     5821 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     5881 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5941 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6001 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6061 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6121 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6181 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6241 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6301 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6361 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6421 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6481 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6541 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6601 ttttgctcac atgtt
//
