LOCUS       sequence-199332-        7035 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 7035)
  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 7035)
  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)-EYFP
FEATURES             Location/Qualifiers
     source          1..7035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   49..178
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        197..1485
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1518..1527
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1524..2450
                     /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"
     regulatory      2460..2469
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2466..2468,2469..2471,2472..3185)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2511..2532)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2772..2791)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3119..3140
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3210..3798
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3263..3283)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3681..3692)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3830..4306
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4029..4048)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4346..4486
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4346..4475
                     /label=AAV2 ITR
     rep_origin      4561..5016
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4648..4667)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4858..4879
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5033..5052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5152..5174
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5212..5230)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5298..5402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5403..5471,5472..6263)
                     /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(5621..5640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6434..7022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6923..6942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc
       61 tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc
      121 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc
      181 ggccgcacgc gtttaacatt atggccttag gtcacttcat ctccatgggg ttcttcttct
      241 gattttctag aaaatgagat gggggtgcag agagcttcct cagtgacctg cccagggtca
      301 catcagaaat gtcagagcta gaacttgaac tcagattact aatcttaaat tccatgcctt
      361 gggggcatgc aagtacgata tacagaagga gtgaactcat tagggcagat gaccaatgag
      421 tttaggaaag aagagtccag ggcagggtac atctacacca cccgcccagc cctgggtgag
      481 tccagccacg ttcacctcat tatagttgcc tctctccagt cctaccttga cgggaagcac
      541 aagcagaaac tgggacagga gccccaggag accaaatctt catggtccct ctgggaggat
      601 gggtggggag agctgtggca gaggcctcag gaggggccct gctgctcagt ggtgacagat
      661 aggggtgaga aagcagacag agtcattccg tcagcattct gggtctgttt ggtacttctt
      721 ctcacgctaa ggtggcggtg tgatatgcac aatggctaaa aagcagggag agctggaaag
      781 aaacaaggac agagacagag gccaagtcaa ccagaccaat tcccagagga agcaaagaaa
      841 ccattacaga gactacaagg gggaagggaa ggagagatga attagcttcc cctgtaaacc
      901 ttagaaccca gctgttgcca gggcaacggg gcaatacctg tctcttcaga ggagatgaag
      961 ttgccagggt aactacatcc tgtctttctc aaggaccatc ccagaatgtg gcacccacta
     1021 gccgttacca tagcaactgc ctctttgccc cacttaatcc catcccgtct gttaaaaggg
     1081 ccctatagtt ggaggtgggg gaggtaggaa gagcgatgat cacttgtgga ctaagtttgt
     1141 tcgcatcccc ttctccaacc ccctcagtac atcaccctgg gggaacaggg tccacttgct
     1201 cctgggccca cacagtcctg cagtattgtg tatataaggc cagggcaaag aggagcaggt
     1261 tttaaagtga aaggcaggca ggtgttgggg aggcagttac cggggcaacg ggaacagggc
     1321 gtttcggagg tggttgccat ggggacctgg atgctgacga aggctcgcga ggctgtgagc
     1381 agccacagtg ccctgctcag aagccccaag ctcgtcagtc aagccggttc tccgtttgca
     1441 ctcaggagca cgggcaggcg agtggcccct agttctgggg gcagctctag agcggtaccg
     1501 gatccccggg taccggtgcc accatggatt atggaggcgc tttgtctgcc gtcggacgcg
     1561 aacttttgtt cgttactaat cctgtggtgg tgaacgggtc cgtcctggtc cctgaggatc
     1621 aatgttactg tgccggatgg attgaatctc gcggcacgaa cggcgctcag accgcgtcaa
     1681 atgtcctgca gtggcttgca gcaggattca gcattttgct gctgatgttc tatgcctacc
     1741 aaacctggaa atctacatgc ggctgggagg agatctatgt gtgcgccatt gaaatggtta
     1801 aggtgattct cgagttcttt tttgagttta agaatccctc tatgctctac cttgccacag
     1861 gacaccgggt gcagtggctg cgctatgcag cctggctgct cacttgtcct gtcatcctta
     1921 tccacctgag caacctcacc ggcctgagca acgactacag caggagaacc atgggactcc
     1981 ttgtctcaga catcgggact atcgtgtggg gggctaccag cgccatggca accggctatg
     2041 ttaaagtcat cttcttttgt cttggattgt gctatggcgc gaacacattt tttcacgccg
     2101 ccaaagcata tatcgagggt tatcatactg tgccaaaggg tcggtgccgc caggtcgtga
     2161 ccggcatggc atggctgttt ttcgtgagct ggggtatgtt cccaattctc ttcattttgg
     2221 ggcccgaagg ttttggcgtc ctgagcgtct atggctccac cgtaggtcac acgattattg
     2281 atctgatgag taaaaattgt tgggggttgt tgggacacta cctgcgcgtc ctgatccacg
     2341 agcacatatt gattcacgga gatatccgca aaaccaccaa actgaacatc ggcggaacgg
     2401 agatcgaggt cgagactctc gtcgaagacg aagccgaggc cggagccgtg ccagcggccg
     2461 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2521 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2581 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2641 ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac cccgaccaca
     2701 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2761 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2821 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2881 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2941 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3001 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3061 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     3121 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3181 agtaagaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     3241 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3301 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3361 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3421 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3481 tttccggaac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3541 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3601 ggaaatcatc gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga
     3661 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3721 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3781 tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc
     3841 ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag
     3901 ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
     3961 tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc
     4021 ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat
     4081 ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca
     4141 ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat
     4201 attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa
     4261 attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt
     4321 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     4381 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4441 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     4501 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     4561 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4621 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4681 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4741 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4801 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4861 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     4921 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     4981 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     5041 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5101 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5161 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     5221 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     5281 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5341 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5401 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5461 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5521 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5581 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5641 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5701 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5761 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5821 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5881 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5941 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6001 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6061 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6121 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6181 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6241 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6301 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6361 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6421 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6481 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6541 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6601 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6661 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6721 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6781 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6841 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6901 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6961 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7021 aaaacgccag caacg
//
