LOCUS       Exported                5757 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector with hSynapsin promoter, Cre-ON ChETA-mRuby2 (for 
            optogenetic activation), and W3SL regulatory cassette (for maximize 
            cloning capacity).
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO {ChETA-mRuby2}on-W3SL
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5757)
  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 5757)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5757
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    660..693
                     /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    744..777
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(790..1500)
                     /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      1497..1506
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(1516..2442)
                     /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      2439..2448
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(2453..2486)
                     /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(2537..2570)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(2640..2660)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3020..3160
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3020..3149
                     /label=AAV2 ITR
     rep_origin      3235..3690
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3322..3341)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3532..3553
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3707..3726)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3826..3848
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3886..3904)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3972..4076
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4077..4937
                     /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(4295..4314)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5108..5696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5597..5616
                     /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 gcgtaattac ttgtacagct cgtccatccc accaccaagc ccggcgaact tggcaactgc
      841 gtgttcgcgt tgtactacga acatttcatt gtcactttcc tctaaccttt ccaggcggtg
      901 atcaacggca tggataccgg gcatcttgat gttcccgacg gtcttttttg acctgtaagt
      961 tgttacgaaa gagcaagaca gatggccacc accatcaact ttcagtgcca tatgagtgta
     1021 tcccctcaga ccaccatctg ctggatacat catctctgta ttaggctccc aacccttggt
     1081 cttcttctgc atcacgggac cattggaggg aaagtttacc cctctgactt ggacgtggta
     1141 aacgagacag ccatcctcaa ggctggtgtc ctgcatgacg gtgacgactc caccatcttc
     1201 gtatctcgta actctttccc aagtaaaacc ctcaggaaag gactgtttaa agaaatcagg
     1261 aatgcctttc gggtacttga taaaagtacg gctgccatac atgaacgacg tggcaagaat
     1321 gtcaaaggca aatggcaggg gtcctccctc gatgactttg atcctcatgg tttgagttcc
     1381 catgtacgga ttgccttctc cttcacctgt gcatttgaat tggtggccgt tgaccgaacc
     1441 ttccatgacc accttcatac gcatattttc cttgatcagc tcttcgccct tagacaccat
     1501 ggtggcggcc gctggcacgg ctccggcctc ggcttcgtct tcgacgagag tctcgacctc
     1561 gatctccgtt ccgccgatgt tcagtttggt ggttttgcgg atatctccgt gaatcaatat
     1621 gtgctcgtgg atcaggacgc gcaggtagtg tcccaacaac ccccaacaat ttttactcat
     1681 cagatcaata atcgtgtgac ctacggtgga gccatagacg ctcaggacgc caaaaccttc
     1741 gggccccaaa atgaagagaa ttgggaacat accccagctc acgaaaaaca gccatgccat
     1801 gccggtcacg acctggcggc accgaccctt tggcacagta tgataaccct cgatatatgc
     1861 tttggcggcg tgaaaaaatg tgttcgcgcc atagcacaat ccaagacaaa agaagatgac
     1921 tttaacatag ccggttgcca tggcgctggt agccccccac acgatagtcc cgatgtctga
     1981 gacaaggagt cccatggttc tcctgctgta gtcgttgctc aggccggtga ggttgctcag
     2041 gcggataagg atgacaggac aagtgagcag ccaggttgca tagcgcagcc actgcacccg
     2101 gtgtcctgtg gcaaggtaga gcatagaggg attcttaaac tcaaaaaaga actcgagaat
     2161 caccttaacc atttcaatgg cgcacacata gatctcctcc cagccgcatg tagatttcca
     2221 ggtttggtag gcatagaaca tcagcagcaa aatgctgaat cctgctgcaa gccactgcag
     2281 gacatttgac gcggtctgag cgccgttcgt gccgcgagat tcaatccatc cggcacagta
     2341 acattgatcc tcagggacca ggacggaccc gttcaccacc acaggattag taacgaacaa
     2401 aagttcgcgt ccgacggcag acaaagcgcc gccatagtcc atggtggcta gcataacttc
     2461 gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt
     2521 tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat gaattccgct
     2581 cgagataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2641 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2701 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttagttct tgccacggcg
     2761 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     2821 aattccgtgg tgtttatttg tgaaatttgt gatgctattg ctttatttgt aaccatctag
     2881 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2941 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga
     3001 ggttttttaa agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     3061 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     3121 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     3181 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     3241 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3301 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3361 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3421 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     3481 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3541 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     3601 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3661 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     3721 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3781 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3841 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3901 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     3961 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4021 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4081 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4141 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4201 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4261 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4321 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4381 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4441 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4501 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4561 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4621 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4681 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4741 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4801 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4861 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4921 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4981 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5221 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
