LOCUS       sequence-194448-        6224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of humanized ChR2 with E123T/H134R mutations fused to
            EYFP driven by hSynap promoter for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6224)
  TITLE     Deisseroth AAV plasmids
REFERENCE   2  (bases 1 to 6224)
  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"
COMMENT     Sequence Label: pAAV.hSynap.ChETA(E123T/H134R)-eYFP.WPRE.hGH
FEATURES             Location/Qualifiers
     source          1..6224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      259..268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             265..1191
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      1201..1210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1207..1209,1210..1212,1213..1926)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1252..1273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1513..1532)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1860..1881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1951..2539
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2004..2024)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2422..2433)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2571..3047
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2770..2789)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3087..3227
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3087..3216
                     /label=AAV2 ITR
     rep_origin      3302..3757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3389..3408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3599..3620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3774..3793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3893..3915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3953..3971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4039..4143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4144..4212,4213..5004)
                     /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(4362..4381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5175..5763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5664..5683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5825..5954
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(5994..6224,1..217)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg
       61 tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg
      121 gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt
      181 gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg aattcaagct gctagcaagg
      241 atcctctaga gtcgactagc caccatggac tatggcggcg ctttgtctgc cgtcggacgc
      301 gaacttttgt tcgttactaa tcctgtggtg gtgaacgggt ccgtcctggt ccctgaggat
      361 caatgttact gtgccggatg gattgaatct cgcggcacga acggcgctca gaccgcgtca
      421 aatgtcctgc agtggcttgc agcaggattc agcattttgc tgctgatgtt ctatgcctac
      481 caaacctgga aatctacatg cggctgggag gagatctatg tgtgcgccat tgaaatggtt
      541 aaggtgattc tcgagttctt ttttgagttt aagaatccct ctatgctcta ccttgccaca
      601 ggacaccggg tgcagtggct gcgctatgca acctggctgc tcacttgtcc tgtcatcctt
      661 atccgcctga gcaacctcac cggcctgagc aacgactaca gcaggagaac catgggactc
      721 cttgtctcag acatcgggac tatcgtgtgg ggggctacca gcgccatggc aaccggctat
      781 gttaaagtca tcttcttttg tcttggattg tgctatggcg cgaacacatt ttttcacgcc
      841 gccaaagcat atatcgaggg ttatcatact gtgccaaagg gtcggtgccg ccaggtcgtg
      901 accggcatgg catggctgtt tttcgtgagc tggggtatgt tcccaattct cttcattttg
      961 gggcccgaag gttttggcgt cctgagcgtc tatggctcca ccgtaggtca cacgattatt
     1021 gatctgatga gtaaaaattg ttgggggttg ttgggacact acctgcgcgt cctgatccac
     1081 gagcacatat tgattcacgg agatatccgc aaaaccacca aactgaacat cggcggaacg
     1141 gagatcgagg tcgagactct cgtcgaagac gaagccgagg ccggagccgt gccagcggcc
     1201 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1261 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1321 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1381 cccaccctcg tgaccacctt cggctacggc ctgcagtgct tcgcccgcta ccccgaccac
     1441 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1501 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1561 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1621 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1681 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1741 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1801 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1861 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1921 aagtaagaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     1981 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2041 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     2101 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2161 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2221 ctttccggaa ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2281 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2341 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     2401 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     2461 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     2521 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     2581 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     2641 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     2701 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     2761 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     2821 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     2881 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     2941 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     3001 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     3061 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     3121 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3181 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3241 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3301 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3361 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3421 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3481 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     3541 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3601 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     3661 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     3721 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     3781 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     3841 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3901 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3961 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4021 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4081 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4141 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4201 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4261 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4321 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4381 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4441 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4501 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4561 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4621 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4681 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4741 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4801 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4861 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4921 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4981 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5041 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5101 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5161 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5221 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5281 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5341 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5401 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5461 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5521 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5581 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5641 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5701 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5761 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5821 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     5881 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     5941 cactaggggt tcctgcggcc gcacgcggat ctctgcagag ggccctgcgt atgagtgcaa
     6001 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac
     6061 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg
     6121 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg
     6181 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcac
//
