LOCUS       Exported                7020 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV expression of CaMKIIa-driven hChR2 variant for ultrafast 
            optogenetic control..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7020)
  AUTHORS   Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R,
            Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K
  TITLE     Principles for applying optogenetic tools derived from direct 
            comparative analysis of microbial opsins.
  JOURNAL   Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808.
  PUBMED    22179551
REFERENCE   2  (bases 1 to 7020)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7020
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   52..181
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        200..1488
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1521..1530
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1527..2453
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             2466..3173
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2608..2626)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2876..2895
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3198..3786
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3251..3271)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3669..3680)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3818..4294
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4334..4474
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4334..4463
                     /label=AAV2 ITR
     rep_origin      4549..5004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4636..4655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4846..4867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5021..5040)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5140..5162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5200..5218)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5286..5390
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5391..6251
                     /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(5609..5628)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6422..7010
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6911..6930
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc
       61 agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg
      121 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc
      181 tgcggccgca cgcgtttaac attatggcct taggtcactt catctccatg gggttcttct
      241 tctgattttc tagaaaatga gatgggggtg cagagagctt cctcagtgac ctgcccaggg
      301 tcacatcaga aatgtcagag ctagaacttg aactcagatt actaatctta aattccatgc
      361 cttgggggca tgcaagtacg atatacagaa ggagtgaact cattagggca gatgaccaat
      421 gagtttagga aagaagagtc cagggcaggg tacatctaca ccacccgccc agccctgggt
      481 gagtccagcc acgttcacct cattatagtt gcctctctcc agtcctacct tgacgggaag
      541 cacaagcaga aactgggaca ggagccccag gagaccaaat cttcatggtc cctctgggag
      601 gatgggtggg gagagctgtg gcagaggcct caggaggggc cctgctgctc agtggtgaca
      661 gataggggtg agaaagcaga cagagtcatt ccgtcagcat tctgggtctg tttggtactt
      721 cttctcacgc taaggtggcg gtgtgatatg cacaatggct aaaaagcagg gagagctgga
      781 aagaaacaag gacagagaca gaggccaagt caaccagacc aattcccaga ggaagcaaag
      841 aaaccattac agagactaca agggggaagg gaaggagaga tgaattagct tcccctgtaa
      901 accttagaac ccagctgttg ccagggcaac ggggcaatac ctgtctcttc agaggagatg
      961 aagttgccag ggtaactaca tcctgtcttt ctcaaggacc atcccagaat gtggcaccca
     1021 ctagccgtta ccatagcaac tgcctctttg ccccacttaa tcccatcccg tctgttaaaa
     1081 gggccctata gttggaggtg ggggaggtag gaagagcgat gatcacttgt ggactaagtt
     1141 tgttcgcatc cccttctcca accccctcag tacatcaccc tgggggaaca gggtccactt
     1201 gctcctgggc ccacacagtc ctgcagtatt gtgtatataa ggccagggca aagaggagca
     1261 ggttttaaag tgaaaggcag gcaggtgttg gggaggcagt taccggggca acgggaacag
     1321 ggcgtttcgg aggtggttgc catggggacc tggatgctga cgaaggctcg cgaggctgtg
     1381 agcagccaca gtgccctgct cagaagcccc aagctcgtca gtcaagccgg ttctccgttt
     1441 gcactcagga gcacgggcag gcgagtggcc cctagttctg ggggcagctc tagagcggta
     1501 ccggatcccc gggtaccggt gccaccatgg actatggcgg cgctttgtct gccgtcggac
     1561 gcgaactttt gttcgttact aatcctgtgg tggtgaacgg gtccgtcctg gtccctgagg
     1621 atcaatgtta ctgtgccgga tggattgaat ctcgcggcac gaacggcgct cagaccgcgt
     1681 caaatgtcct gcagtggctt gcagcaggat tcagcatttt gctgctgatg ttctatgcct
     1741 accaaacctg gaaatctaca tgcggctggg aggagatcta tgtgtgcgcc attgaaatgg
     1801 ttaaggtgat tctcgagttc ttttttgagt ttaagaatcc ctctatgctc taccttgcca
     1861 caggacaccg ggtgcagtgg ctgcgctatg caacctggct gctcacttgt cctgtcatcc
     1921 ttatccacct gagcaacctc accggcctga gcaacgacta cagcaggaga accatgggac
     1981 tccttgtctc agacatcggg tgtatcgtgt ggggggctac cagcgccatg gcaaccggct
     2041 atgttaaagt catcttcttt tgtcttggat tgtgctatgg cgcgaacaca ttttttcacg
     2101 ccgccaaagc atatatcgag ggttatcata ctgtgccaaa gggtcggtgc cgccaggtcg
     2161 tgaccggcat ggcatggctg tttttcgtga gctggggtat gttcccaatt ctcttcattt
     2221 tggggcccga aggttttggc gtcctgagcg tctatggctc caccgtaggt cacacgatta
     2281 ttgatctgat gagtaaaaat tgttgggggt tgttgggaca ctacctgcgc gtcctgatcc
     2341 acgagcacat attgattcac ggagatatcc gcaaaaccac caaactgaac atcggcggaa
     2401 cggagatcga ggtcgagact ctcgtcgaag acgaagccga ggccggagcc gtgccagcgg
     2461 ccgccgtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     2521 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2581 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2641 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     2701 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2761 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2821 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     2881 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     2941 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     3001 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     3061 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     3121 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg
     3181 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3241 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3301 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3361 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3421 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccggaactt
     3481 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3541 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3601 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct
     3661 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     3721 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     3781 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc
     3841 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat
     3901 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag
     3961 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta
     4021 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
     4081 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac
     4141 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct
     4201 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt
     4261 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg
     4321 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4441 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4501 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4621 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4741 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4861 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     4921 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     4981 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5041 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5101 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5161 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5221 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5461 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5521 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5581 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5641 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5701 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5761 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5821 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5881 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5941 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6001 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6061 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6121 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6181 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6241 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6301 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6361 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6421 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6481 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6541 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6601 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6661 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6721 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6781 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6841 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6901 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6961 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
//
