LOCUS       sequence-200275-        7020 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of CaMKIIa-driven hChR2 variant CheTA 2.0 for 
            ultrafast optogenetic control..
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2011-12-18- 18"; volume: "9"; 
            issue: "2"; pages: "159-72"
COMMENT     Sequence Label: pAAV-CaMKIIa-hChR2(E123A)-mCherry
FEATURES             Location/Qualifiers
     source          1..7020
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   76..216
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   76..205
                     /label=AAV2 ITR
     rep_origin      291..746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(378..397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(763..782)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     882..904
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(942..960)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1028..1132
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1133..1201,1202..1993)
                     /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(1351..1370)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2164..2752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2653..2672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2814..2943
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2962..4250
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      4283..4292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4289..5215
                     /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             5228..5935
                     /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(5370..5388)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5638..5657
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    5960..6548
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6013..6033)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6431..6442)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    join(6580..7020,1..36)
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(6779..6798)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
ORIGIN
        1 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
       61 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
      121 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      181 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
      241 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
      301 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      361 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      421 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      481 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
      541 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      601 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
      661 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      721 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
      781 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      841 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      901 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
      961 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     1021 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1081 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1141 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1201 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1261 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1321 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1381 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1441 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1501 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1561 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1621 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1681 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1741 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1801 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     1861 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1921 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1981 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     2041 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2101 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2161 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2221 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2281 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2341 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2401 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2461 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2521 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2581 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2641 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2701 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2761 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     2821 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
     2881 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     2941 cctgcggccg cacgcgttta acattatggc cttaggtcac ttcatctcca tggggttctt
     3001 cttctgattt tctagaaaat gagatggggg tgcagagagc ttcctcagtg acctgcccag
     3061 ggtcacatca gaaatgtcag agctagaact tgaactcaga ttactaatct taaattccat
     3121 gccttggggg catgcaagta cgatatacag aaggagtgaa ctcattaggg cagatgacca
     3181 atgagtttag gaaagaagag tccagggcag ggtacatcta caccacccgc ccagccctgg
     3241 gtgagtccag ccacgttcac ctcattatag ttgcctctct ccagtcctac cttgacggga
     3301 agcacaagca gaaactggga caggagcccc aggagaccaa atcttcatgg tccctctggg
     3361 aggatgggtg gggagagctg tggcagaggc ctcaggaggg gccctgctgc tcagtggtga
     3421 cagatagggg tgagaaagca gacagagtca ttccgtcagc attctgggtc tgtttggtac
     3481 ttcttctcac gctaaggtgg cggtgtgata tgcacaatgg ctaaaaagca gggagagctg
     3541 gaaagaaaca aggacagaga cagaggccaa gtcaaccaga ccaattccca gaggaagcaa
     3601 agaaaccatt acagagacta caagggggaa gggaaggaga gatgaattag cttcccctgt
     3661 aaaccttaga acccagctgt tgccagggca acggggcaat acctgtctct tcagaggaga
     3721 tgaagttgcc agggtaacta catcctgtct ttctcaagga ccatcccaga atgtggcacc
     3781 cactagccgt taccatagca actgcctctt tgccccactt aatcccatcc cgtctgttaa
     3841 aagggcccta tagttggagg tgggggaggt aggaagagcg atgatcactt gtggactaag
     3901 tttgttcgca tccccttctc caaccccctc agtacatcac cctgggggaa cagggtccac
     3961 ttgctcctgg gcccacacag tcctgcagta ttgtgtatat aaggccaggg caaagaggag
     4021 caggttttaa agtgaaaggc aggcaggtgt tggggaggca gttaccgggg caacgggaac
     4081 agggcgtttc ggaggtggtt gccatgggga cctggatgct gacgaaggct cgcgaggctg
     4141 tgagcagcca cagtgccctg ctcagaagcc ccaagctcgt cagtcaagcc ggttctccgt
     4201 ttgcactcag gagcacgggc aggcgagtgg cccctagttc tgggggcagc tctagagcgg
     4261 taccggatcc ccgggtaccg gtgccaccat ggattatgga ggcgctttgt ctgccgtcgg
     4321 acgcgaactt ttgttcgtta ctaatcctgt ggtggtgaac gggtccgtcc tggtccctga
     4381 ggatcaatgt tactgtgccg gatggattga atctcgcggc acgaacggcg ctcagaccgc
     4441 gtcaaatgtc ctgcagtggc ttgcagcagg attcagcatt ttgctgctga tgttctatgc
     4501 ctaccaaacc tggaaatcta catgcggctg ggaggagatc tatgtgtgcg ccattgaaat
     4561 ggttaaggtg attctcgagt tcttttttga gtttaagaat ccctctatgc tctaccttgc
     4621 cacaggacac cgggtgcagt ggctgcgcta tgcagcctgg ctgctcactt gtcctgtcat
     4681 ccttatccac ctgagcaacc tcaccggcct gagcaacgac tacagcagga gaaccatggg
     4741 actccttgtc tcagacatcg ggactatcgt gtggggggct accagcgcca tggcaaccgg
     4801 ctatgttaaa gtcatcttct tttgtcttgg attgtgctat ggcgcgaaca cattttttca
     4861 cgccgccaaa gcatatatcg agggttatca tactgtgcca aagggtcggt gccgccaggt
     4921 cgtgaccggc atggcatggc tgtttttcgt gagctggggt atgttcccaa ttctcttcat
     4981 tttggggccc gaaggttttg gcgtcctgag cgtctatggc tccaccgtag gtcacacgat
     5041 tattgatctg atgagtaaaa attgttgggg gttgttggga cactacctgc gcgtcctgat
     5101 ccacgagcac atattgattc acggagatat ccgcaaaacc accaaactga acatcggcgg
     5161 aacggagatc gaggtcgaga ctctcgtcga agacgaagcc gaggccggag ccgtgccagc
     5221 ggccgccgtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     5281 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     5341 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     5401 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     5461 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     5521 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     5581 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     5641 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     5701 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     5761 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     5821 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     5881 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagaatt
     5941 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     6001 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     6061 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     6121 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     6181 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccggaac
     6241 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     6301 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     6361 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     6421 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     6481 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     6541 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     6601 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     6661 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     6721 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     6781 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     6841 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     6901 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     6961 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
//
