LOCUS       Exported                6015 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV production of CheRiff channelrhodopsin actuator.
ACCESSION   .
VERSION     .
KEYWORDS    DRH337: AAV-hsyn-CheRiff-eGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6015)
  AUTHORS   Hochbaum DR, Zhao Y, Farhi SL, Klapoetke N, Werley CA, Kapoor V, Zou
            P, Kralj JM, Maclaurin D, Smedemark-Margulies N, Saulnier JL, 
            Boulting GL, Straub C, Cho YK, Melkonian M, Wong GK, Harrison DJ, 
            Murthy VN, Sabatini BL, Boyden ES, Campbell RE, Cohen AE
  TITLE     All-optical electrophysiology in mammalian neurons using engineered 
            microbial rhodopsins.
  JOURNAL   Nat Methods. 2014 Aug;11(8):825-33. doi: 10.1038/nmeth.3000. Epub 
            2014 Jun 22.
  PUBMED    24952910
REFERENCE   2  (bases 1 to 6015)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6015
                     /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        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      642..651
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             648..1595
                     /codon_start=1
                     /product="blue light-activated optogenetic actuator derived
                     from Scherffelia dubia channelrhodopsin (Hochbaum et al., 
                     2014)"
                     /label=CheRiff
                     /translation="MGGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQ
                     CSSIYIYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVELIKV
                     ILEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITGLSEAYNKRTMALL
                     VSDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFYNAGIIYVESYYIMPAGGCKKLV
                     LAMTAVYYSSWLMFPGLFIFGPEGMHTLSVAGSTIGHTIADLLSKNIWGLLGHFLRIKI
                     HEHIIMYGDIRRPVSSQFLGRKVDVLAFVTEEDKV"
     CDS             1665..2378
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1707..1728)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1968..1987)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2315..2336
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2406..2994
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2459..2479)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2877..2888)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3036..3053)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3042..3249
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3278..3418
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3278..3407
                     /label=AAV2 ITR
     rep_origin      3493..3948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3580..3599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3790..3811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3965..3984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4084..4106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4144..4162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4230..4334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4335..5195
                     /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(4553..4572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5366..5954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5855..5874
                     /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 gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg ggcggagctc
      661 ctgctccaga cgctcacagc gccccacctg gaaacgattc tgccggaggc agtgagtacc
      721 atgccccagc tggatatcaa gtgaatccac cctaccaccc cgtgcatggg tatgaggaac
      781 agtgcagctc catctacatc tactatgggg ccctgtggga gcaggaaaca gctaggggct
      841 tccagtggtt tgccgtgttc ctgtctgccc tgtttctggc tttctacggc tggcacgcct
      901 ataaggccag cgtgggatgg gaggaagtgt acgtgtgctc cgtggagctg atcaaagtga
      961 ttctggagat ctatttcgag ttcaccagtc ctgctatgct gttcctgtac ggagggaaca
     1021 ttaccccatg gctgagatat gccgaatggc tgctgacatg tcccgtgatc ctgattcatc
     1081 tgtctaacat caccggcctg agtgaggcat acaataagcg gacaatggct ctgctggtgt
     1141 ccgacctggg aactatttgc atgggagtga cagccgctct ggccactggg tgggtgaagt
     1201 ggctgtttta ctgtatcggc ctggtgtatg gaacccagac attctacaac gctggaatca
     1261 tctacgtgga gtcttactat atcatgcctg ccggcggctg taagaaactg gtgctggcca
     1321 tgactgccgt gtactattct agttggctga tgtttcccgg cctgttcatc tttgggcctg
     1381 aaggcatgca caccctgagc gtggctgggt ccactattgg ccataccatc gccgacctgc
     1441 tgtccaagaa tatttgggga ctgctggggc acttcctgcg gatcaaaatt cacgagcata
     1501 tcattatgta cggcgatatc aggagaccag tgagctccca gtttctggga cgcaaggtgg
     1561 acgtgctggc cttcgtgaca gaggaagata aagtggcggc cgccaagagc aggatcacca
     1621 gcgagggcga gtacatcccc ctggaccaga tcgacatcaa cgtggtgagc aagggcgagg
     1681 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1741 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1801 tcatttgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1861 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1921 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1981 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2041 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2101 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2161 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2221 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     2281 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2341 ccgccgggat cactctcggc atggacgagc tgtacaagta agaattcgat atcaagctta
     2401 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2461 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     2521 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     2581 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     2641 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     2701 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     2761 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     2821 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     2881 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     2941 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     3001 accgagcgct gctcgagcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     3061 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3121 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3181 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3241 tgctggggag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3301 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3361 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3481 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3541 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3781 tctttaatag tggactcttg ttccaaactg gaacaacact caatcctatc tcgggctatt
     3841 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     3901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3961 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4141 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4201 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5041 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6001 cttttgctca catgt
//
