LOCUS       Exported                7020 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV expression of CaMKIIa-driven hChR2 variant CheTA 2.0 for 
            ultrafast optogenetic control..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CaMKIIa-hChR2(E123A)-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   54..183
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        202..1490
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1523..1532
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1529..2455
                     /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
                     PSMLYLATGHRVQWLRYAAWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             2468..3175
                     /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(2610..2628)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2878..2897
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3200..3788
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3253..3273)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3671..3682)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3820..4296
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4019..4038)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4336..4476
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4336..4465
                     /label=AAV2 ITR
     rep_origin      4551..5006
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4638..4657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4848..4869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5023..5042)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5142..5164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5202..5220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5288..5392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5393..6253
                     /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(5611..5630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6424..7012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6913..6932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
       61 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
      121 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      181 cctgcggccg cacgcgttta acattatggc cttaggtcac ttcatctcca tggggttctt
      241 cttctgattt tctagaaaat gagatggggg tgcagagagc ttcctcagtg acctgcccag
      301 ggtcacatca gaaatgtcag agctagaact tgaactcaga ttactaatct taaattccat
      361 gccttggggg catgcaagta cgatatacag aaggagtgaa ctcattaggg cagatgacca
      421 atgagtttag gaaagaagag tccagggcag ggtacatcta caccacccgc ccagccctgg
      481 gtgagtccag ccacgttcac ctcattatag ttgcctctct ccagtcctac cttgacggga
      541 agcacaagca gaaactggga caggagcccc aggagaccaa atcttcatgg tccctctggg
      601 aggatgggtg gggagagctg tggcagaggc ctcaggaggg gccctgctgc tcagtggtga
      661 cagatagggg tgagaaagca gacagagtca ttccgtcagc attctgggtc tgtttggtac
      721 ttcttctcac gctaaggtgg cggtgtgata tgcacaatgg ctaaaaagca gggagagctg
      781 gaaagaaaca aggacagaga cagaggccaa gtcaaccaga ccaattccca gaggaagcaa
      841 agaaaccatt acagagacta caagggggaa gggaaggaga gatgaattag cttcccctgt
      901 aaaccttaga acccagctgt tgccagggca acggggcaat acctgtctct tcagaggaga
      961 tgaagttgcc agggtaacta catcctgtct ttctcaagga ccatcccaga atgtggcacc
     1021 cactagccgt taccatagca actgcctctt tgccccactt aatcccatcc cgtctgttaa
     1081 aagggcccta tagttggagg tgggggaggt aggaagagcg atgatcactt gtggactaag
     1141 tttgttcgca tccccttctc caaccccctc agtacatcac cctgggggaa cagggtccac
     1201 ttgctcctgg gcccacacag tcctgcagta ttgtgtatat aaggccaggg caaagaggag
     1261 caggttttaa agtgaaaggc aggcaggtgt tggggaggca gttaccgggg caacgggaac
     1321 agggcgtttc ggaggtggtt gccatgggga cctggatgct gacgaaggct cgcgaggctg
     1381 tgagcagcca cagtgccctg ctcagaagcc ccaagctcgt cagtcaagcc ggttctccgt
     1441 ttgcactcag gagcacgggc aggcgagtgg cccctagttc tgggggcagc tctagagcgg
     1501 taccggatcc ccgggtaccg gtgccaccat ggattatgga ggcgctttgt ctgccgtcgg
     1561 acgcgaactt ttgttcgtta ctaatcctgt ggtggtgaac gggtccgtcc tggtccctga
     1621 ggatcaatgt tactgtgccg gatggattga atctcgcggc acgaacggcg ctcagaccgc
     1681 gtcaaatgtc ctgcagtggc ttgcagcagg attcagcatt ttgctgctga tgttctatgc
     1741 ctaccaaacc tggaaatcta catgcggctg ggaggagatc tatgtgtgcg ccattgaaat
     1801 ggttaaggtg attctcgagt tcttttttga gtttaagaat ccctctatgc tctaccttgc
     1861 cacaggacac cgggtgcagt ggctgcgcta tgcagcctgg ctgctcactt gtcctgtcat
     1921 ccttatccac ctgagcaacc tcaccggcct gagcaacgac tacagcagga gaaccatggg
     1981 actccttgtc tcagacatcg ggactatcgt gtggggggct accagcgcca tggcaaccgg
     2041 ctatgttaaa gtcatcttct tttgtcttgg attgtgctat ggcgcgaaca cattttttca
     2101 cgccgccaaa gcatatatcg agggttatca tactgtgcca aagggtcggt gccgccaggt
     2161 cgtgaccggc atggcatggc tgtttttcgt gagctggggt atgttcccaa ttctcttcat
     2221 tttggggccc gaaggttttg gcgtcctgag cgtctatggc tccaccgtag gtcacacgat
     2281 tattgatctg atgagtaaaa attgttgggg gttgttggga cactacctgc gcgtcctgat
     2341 ccacgagcac atattgattc acggagatat ccgcaaaacc accaaactga acatcggcgg
     2401 aacggagatc gaggtcgaga ctctcgtcga agacgaagcc gaggccggag ccgtgccagc
     2461 ggccgccgtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     2521 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     2581 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     2641 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     2701 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     2761 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     2821 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     2881 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     2941 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     3001 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     3061 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     3121 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagaatt
     3181 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3241 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3301 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3361 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3421 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccggaac
     3481 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3541 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3601 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3661 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3721 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3781 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     3841 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     3901 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     3961 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4021 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4081 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4141 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4201 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4261 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4321 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4381 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4441 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4501 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4561 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4621 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4681 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4741 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4801 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4861 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     4921 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4981 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5041 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5101 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5161 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5221 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5281 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5341 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5401 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5461 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5521 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5581 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5641 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5701 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5761 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5821 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5881 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5941 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6001 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6061 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6121 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6181 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6241 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6301 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6361 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6421 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6481 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6541 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6601 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6661 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6721 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6781 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6841 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6901 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6961 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
//
