LOCUS       sequence-199313-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of humanized ChR2 with H134R mutation fused to EYFP 
            driven by human Synapsin I promoter for optogenetic excitation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  TITLE     hChR2 plasmids
REFERENCE   2  (bases 1 to 6225)
  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-hSyn-hChR2(H134R)-EYFP
FEATURES             Location/Qualifiers
     source          1..6225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      450..459
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             456..1382
                     /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      1392..1401
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1398..1400,1401..1403,1404..2117)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1443..1464)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1704..1723)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2051..2072
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2142..2730
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2195..2215)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2613..2624)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2762..3238
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2961..2980)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3278..3418
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3278..3407
                     /label=AAV2 ITR
     rep_origin      3493..3948
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3580..3599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3790..3811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3965..3984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4084..4106
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4144..4162)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4230..4334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4335..4403,4404..5195)
                     /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(4553..4572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5366..5954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5855..5874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6016..6145
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(6190..6225,1..412)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 tgggggtgcc tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc
       61 ccaaattgcg catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg
      121 cgcactgcca gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca
      181 ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct
      241 tcccggccac cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag
      301 gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct
      361 gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agtcgagaag
      421 gtaccggatc ctctagagtc gactccggag ccaccatgga ctatggcggc gctttgtctg
      481 ccgtcggacg cgaacttttg ttcgttacta atcctgtggt ggtgaacggg tccgtcctgg
      541 tccctgagga tcaatgttac tgtgccggat ggattgaatc tcgcggcacg aacggcgctc
      601 agaccgcgtc aaatgtcctg cagtggcttg cagcaggatt cagcattttg ctgctgatgt
      661 tctatgccta ccaaacctgg aaatctacat gcggctggga ggagatctat gtgtgcgcca
      721 ttgaaatggt taaggtgatt ctcgagttct tttttgagtt taagaatccc tctatgctct
      781 accttgccac aggacaccgg gtgcagtggc tgcgctatgc agagtggctg ctcacttgtc
      841 ctgtcatcct tatccgcctg agcaacctca ccggcctgag caacgactac agcaggagaa
      901 ccatgggact ccttgtctca gacatcggga ctatcgtgtg gggggctacc agcgccatgg
      961 caaccggcta tgttaaagtc atcttctttt gtcttggatt gtgctatggc gcgaacacat
     1021 tttttcacgc cgccaaagca tatatcgagg gttatcatac tgtgccaaag ggtcggtgcc
     1081 gccaggtcgt gaccggcatg gcatggctgt ttttcgtgag ctggggtatg ttcccaattc
     1141 tcttcatttt ggggcccgaa ggttttggcg tcctgagcgt ctatggctcc accgtaggtc
     1201 acacgattat tgatctgatg agtaaaaatt gttgggggtt gttgggacac tacctgcgcg
     1261 tcctgatcca cgagcacata ttgattcacg gagatatccg caaaaccacc aaactgaaca
     1321 tcggcggaac ggagatcgag gtcgagactc tcgtcgaaga cgaagccgag gccggagccg
     1381 tgccagcggc cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1441 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1501 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1561 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct
     1621 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1681 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1741 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1801 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1861 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1921 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1981 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga
     2041 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2101 acgagctgta caagtaagaa ttcgatatca agcttatcga taatcaacct ctggattaca
     2161 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     2221 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     2281 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     2341 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     2401 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     2461 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     2521 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga
     2581 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     2641 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     2701 gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcta
     2761 cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
     2821 agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc
     2881 cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac
     2941 aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg
     3001 gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc ctcccgagtt
     3061 gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg
     3121 gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
     3181 ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct
     3241 gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3301 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3361 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3481 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3541 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     3601 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3661 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3721 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3781 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     3841 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     3901 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3961 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     4021 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4081 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     4141 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4201 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6001 cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
     6061 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     6121 gccaactcca tcactagggg ttcctgcggc cgcacgcgtg tgtctagact gcagagggcc
     6181 ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cgggg
//
