LOCUS       sequence-199310-        7041 bp    DNA     circular SYN 14-APR-2025
DEFINITION  AAV expression of humanized ChR2(H134R) fused to EYFP driven by 
            CaMKIIa promoter for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7041)
  AUTHORS   Lee JH, Durand R, Gradinaru V, Zhang F, Goshen I, Kim DS, Fenno LE, 
            Ramakrishnan C, Deisseroth K
  TITLE     Global and local fMRI signals driven by neurons defined 
            optogenetically by type and wiring.
  JOURNAL   Nature. 2010 Jun 10. 465(7299):788-92.
  PUBMED    20473285
REFERENCE   2  (bases 1 to 7041)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2010 Jun 10. 465(7299):788-92."
COMMENT     Sequence Label: pAAV-CaMKIIa-hChR2(H134R)-EYFP
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   71..200
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        219..1507
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1546..1555
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1552..2478
                     /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
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2488..2497
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2494..2496,2497..2499,2500..3213)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2539..2560)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2800..2819)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3147..3168
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3238..3826
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3291..3311)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3709..3720)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3858..4334
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4057..4076)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4374..4514
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4374..4503
                     /label=AAV2 ITR
     rep_origin      4589..5044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4676..4695)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4886..4907
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5061..5080)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5180..5202
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5240..5258)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5326..5430
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5431..5499,5500..6291)
                     /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(5649..5668)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6462..7041,1..9)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6951..6970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
       61 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg
      121 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
      181 ctccatcact aggggttcct gcggccgcac gcgtttaaca ttatggcctt aggtcacttc
      241 atctccatgg ggttcttctt ctgattttct agaaaatgag atgggggtgc agagagcttc
      301 ctcagtgacc tgcccagggt cacatcagaa atgtcagagc tagaacttga actcagatta
      361 ctaatcttaa attccatgcc ttgggggcat gcaagtacga tatacagaag gagtgaactc
      421 attagggcag atgaccaatg agtttaggaa agaagagtcc agggcagggt acatctacac
      481 cacccgccca gccctgggtg agtccagcca cgttcacctc attatagttg cctctctcca
      541 gtcctacctt gacgggaagc acaagcagaa actgggacag gagccccagg agaccaaatc
      601 ttcatggtcc ctctgggagg atgggtgggg agagctgtgg cagaggcctc aggaggggcc
      661 ctgctgctca gtggtgacag ataggggtga gaaagcagac agagtcattc cgtcagcatt
      721 ctgggtctgt ttggtacttc ttctcacgct aaggtggcgg tgtgatatgc acaatggcta
      781 aaaagcaggg agagctggaa agaaacaagg acagagacag aggccaagtc aaccagacca
      841 attcccagag gaagcaaaga aaccattaca gagactacaa gggggaaggg aaggagagat
      901 gaattagctt cccctgtaaa ccttagaacc cagctgttgc cagggcaacg gggcaatacc
      961 tgtctcttca gaggagatga agttgccagg gtaactacat cctgtctttc tcaaggacca
     1021 tcccagaatg tggcacccac tagccgttac catagcaact gcctctttgc cccacttaat
     1081 cccatcccgt ctgttaaaag ggccctatag ttggaggtgg gggaggtagg aagagcgatg
     1141 atcacttgtg gactaagttt gttcgcatcc ccttctccaa ccccctcagt acatcaccct
     1201 gggggaacag ggtccacttg ctcctgggcc cacacagtcc tgcagtattg tgtatataag
     1261 gccagggcaa agaggagcag gttttaaagt gaaaggcagg caggtgttgg ggaggcagtt
     1321 accggggcaa cgggaacagg gcgtttcgga ggtggttgcc atggggacct ggatgctgac
     1381 gaaggctcgc gaggctgtga gcagccacag tgccctgctc agaagcccca agctcgtcag
     1441 tcaagccggt tctccgtttg cactcaggag cacgggcagg cgagtggccc ctagttctgg
     1501 gggcagctct agagcggtac cggatcctct agagtcgact ccggagccac catggactat
     1561 ggcggcgctt tgtctgccgt cggacgcgaa cttttgttcg ttactaatcc tgtggtggtg
     1621 aacgggtccg tcctggtccc tgaggatcaa tgttactgtg ccggatggat tgaatctcgc
     1681 ggcacgaacg gcgctcagac cgcgtcaaat gtcctgcagt ggcttgcagc aggattcagc
     1741 attttgctgc tgatgttcta tgcctaccaa acctggaaat ctacatgcgg ctgggaggag
     1801 atctatgtgt gcgccattga aatggttaag gtgattctcg agttcttttt tgagtttaag
     1861 aatccctcta tgctctacct tgccacagga caccgggtgc agtggctgcg ctatgcagag
     1921 tggctgctca cttgtcctgt catccttatc cgcctgagca acctcaccgg cctgagcaac
     1981 gactacagca ggagaaccat gggactcctt gtctcagaca tcgggactat cgtgtggggg
     2041 gctaccagcg ccatggcaac cggctatgtt aaagtcatct tcttttgtct tggattgtgc
     2101 tatggcgcga acacattttt tcacgccgcc aaagcatata tcgagggtta tcatactgtg
     2161 ccaaagggtc ggtgccgcca ggtcgtgacc ggcatggcat ggctgttttt cgtgagctgg
     2221 ggtatgttcc caattctctt cattttgggg cccgaaggtt ttggcgtcct gagcgtctat
     2281 ggctccaccg taggtcacac gattattgat ctgatgagta aaaattgttg ggggttgttg
     2341 ggacactacc tgcgcgtcct gatccacgag cacatattga ttcacggaga tatccgcaaa
     2401 accaccaaac tgaacatcgg cggaacggag atcgaggtcg agactctcgt cgaagacgaa
     2461 gccgaggccg gagccgtgcc agcggccgcc accatggtga gcaagggcga ggagctgttc
     2521 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2581 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2641 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcctg
     2701 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2761 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2821 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2881 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2941 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3001 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3061 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc
     3121 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3181 atcactctcg gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat
     3241 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3301 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3361 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3421 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3481 tggggcattg ccaccacctg tcagctcctt tccggaactt tcgctttccc cctccctatt
     3541 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3601 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3661 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3721 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3781 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg
     3841 ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc
     3901 tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt
     3961 tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg
     4021 ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg
     4081 cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc
     4141 ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt
     4201 ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc
     4261 aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc
     4321 ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc
     4381 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4441 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4501 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4561 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     4621 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     4681 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4741 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4801 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4861 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4921 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     4981 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5041 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     5101 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5161 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5221 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     5281 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5341 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5401 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5461 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5521 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5581 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5641 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5701 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5761 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5821 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5881 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5941 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6001 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6061 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6121 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6181 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6241 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6301 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6361 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6601 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7021 tgctcgtcag gggggcggag c
//
