LOCUS       Exported                7046 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)-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7046)
  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 7046)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7046
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        160..1448
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1481..1490
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1487..2413
                     /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      2423..2432
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2429..3148
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2474..2495)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2735..2754)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3082..3103
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3173..3761
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3226..3246)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3644..3655)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3793..4269
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3992..4011)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4309..4449
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4309..4438
                     /label=AAV2 ITR
     rep_origin      4524..4979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4611..4630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4821..4842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4996..5015)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5115..5137
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5175..5193)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5261..5365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5366..6226
                     /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(5584..5603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6397..6985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6886..6905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtttaac attatggcct taggtcactt
      181 catctccatg gggttcttct tctgattttc tagaaaatga gatgggggtg cagagagctt
      241 cctcagtgac ctgcccaggg tcacatcaga aatgtcagag ctagaacttg aactcagatt
      301 actaatctta aattccatgc cttgggggca tgcaagtacg atatacagaa ggagtgaact
      361 cattagggca gatgaccaat gagtttagga aagaagagtc cagggcaggg tacatctaca
      421 ccacccgccc agccctgggt gagtccagcc acgttcacct cattatagtt gcctctctcc
      481 agtcctacct tgacgggaag cacaagcaga aactgggaca ggagccccag gagaccaaat
      541 cttcatggtc cctctgggag gatgggtggg gagagctgtg gcagaggcct caggaggggc
      601 cctgctgctc agtggtgaca gataggggtg agaaagcaga cagagtcatt ccgtcagcat
      661 tctgggtctg tttggtactt cttctcacgc taaggtggcg gtgtgatatg cacaatggct
      721 aaaaagcagg gagagctgga aagaaacaag gacagagaca gaggccaagt caaccagacc
      781 aattcccaga ggaagcaaag aaaccattac agagactaca agggggaagg gaaggagaga
      841 tgaattagct tcccctgtaa accttagaac ccagctgttg ccagggcaac ggggcaatac
      901 ctgtctcttc agaggagatg aagttgccag ggtaactaca tcctgtcttt ctcaaggacc
      961 atcccagaat gtggcaccca ctagccgtta ccatagcaac tgcctctttg ccccacttaa
     1021 tcccatcccg tctgttaaaa gggccctata gttggaggtg ggggaggtag gaagagcgat
     1081 gatcacttgt ggactaagtt tgttcgcatc cccttctcca accccctcag tacatcaccc
     1141 tgggggaaca gggtccactt gctcctgggc ccacacagtc ctgcagtatt gtgtatataa
     1201 ggccagggca aagaggagca ggttttaaag tgaaaggcag gcaggtgttg gggaggcagt
     1261 taccggggca acgggaacag ggcgtttcgg aggtggttgc catggggacc tggatgctga
     1321 cgaaggctcg cgaggctgtg agcagccaca gtgccctgct cagaagcccc aagctcgtca
     1381 gtcaagccgg ttctccgttt gcactcagga gcacgggcag gcgagtggcc cctagttctg
     1441 ggggcagctc tagagcggta ccggatcccc gggtaccggt gccaccatgg attatggagg
     1501 cgctttgtct gccgtcggac gcgaactttt gttcgttact aatcctgtgg tggtgaacgg
     1561 gtccgtcctg gtccctgagg atcaatgtta ctgtgccgga tggattgaat ctcgcggcac
     1621 gaacggcgct cagaccgcgt caaatgtcct gcagtggctt gcagcaggat tcagcatttt
     1681 gctgctgatg ttctatgcct accaaacctg gaaatctaca tgcggctggg aggagatcta
     1741 tgtgtgcgcc attgaaatgg ttaaggtgat tctcgagttc ttttttgagt ttaagaatcc
     1801 ctctatgctc taccttgcca caggacaccg ggtgcagtgg ctgcgctatg cagcctggct
     1861 gctcacttgt cctgtcatcc ttatccacct gagcaacctc accggcctga gcaacgacta
     1921 cagcaggaga accatgggac tccttgtctc agacatcggg actatcgtgt ggggggctac
     1981 cagcgccatg gcaaccggct atgttaaagt catcttcttt tgtcttggat tgtgctatgg
     2041 cgcgaacaca ttttttcacg ccgccaaagc atatatcgag ggttatcata ctgtgccaaa
     2101 gggtcggtgc cgccaggtcg tgaccggcat ggcatggctg tttttcgtga gctggggtat
     2161 gttcccaatt ctcttcattt tggggcccga aggttttggc gtcctgagcg tctatggctc
     2221 caccgtaggt cacacgatta ttgatctgat gagtaaaaat tgttgggggt tgttgggaca
     2281 ctacctgcgc gtcctgatcc acgagcacat attgattcac ggagatatcc gcaaaaccac
     2341 caaactgaac atcggcggaa cggagatcga ggtcgagact ctcgtcgaag acgaagccga
     2401 ggccggagcc gtgccagcgg ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
     2461 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2521 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2581 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg
     2641 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2701 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2761 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2821 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     2881 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     2941 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3001 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga
     3061 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3121 tctcggcatg gacgagctgt acaagtaaga attcgatatc aagcttatcg ataatcaacc
     3181 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3241 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3301 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3361 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3421 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3481 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3541 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     3601 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3661 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3721 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3781 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     3841 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     3901 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     3961 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     4021 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     4081 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     4141 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     4201 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     4261 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag
     4321 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4381 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4441 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4501 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4561 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4621 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4681 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4741 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4801 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4861 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4921 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4981 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5041 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5101 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5161 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5221 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5281 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5341 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5401 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5461 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5521 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5581 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5641 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5701 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5761 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5821 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5881 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5941 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6001 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6061 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6121 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6181 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6241 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6301 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6361 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6421 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6481 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6541 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6601 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6661 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6721 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6781 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6841 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6901 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6961 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7021 cttttgctgg ccttttgctc acatgt
//
