LOCUS       sequence-466066-        6222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Direct-expressing ChR2(H134R)-EYFP AAV virus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6222)
  AUTHORS   Kussick E, Johansen N, Taskin N, Chowdhury A, Quinlan MA, Fraser A, 
            Clark AG, Wynalda B, Martinez R, Groce EL, Reding M, Liang E, Shulga
            L, Huang C, Casper T, Clark M, Ho W, Gao Y, van Velthoven CTJ, 
            Sobieski C, Ferrer R, Berg MR, Curtis BC, English C, Day JC, Fortuna
            MG, Donadio N, Newman D, Yao S, Chakka AB, Goldy J, Torkelson A, 
            Guzman JB, Chakrabarty R, Nguy B, Guilford N, Pham TH, Wright V, 
            Ronellenfitch K, Naidoo R, Kenney J, Williford A, Ramakrishnan C, 
            Drinnenberg A, Gudsnuk K, Thyagarajan B, Smith KA, Dee N, Deisseroth
            K, Zeng H, Yao Z, Tasic B, Levi BP, Hodge R, Bakken TE, Lein ES, 
            Ting JT, Daigle TL
  TITLE     Enhancer AAVs for targeting spinal motor neurons and descending 
            motor pathways in rodents and macaque.
  JOURNAL   Cell Rep. 2025 Jun 24;44(6):115730. doi: 
            10.1016/j.celrep.2025.115730. Epub 2025 May 21.
  PUBMED    40403722
REFERENCE   2  (bases 1 to 6222)
  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"; journalName: "Cell Rep.";
            date: "2025-06-24"; volume: "44(6):115730. doi"; pages: " 
            10.1016/j.celrep.2025.115730. Epub 2025 May 21"
COMMENT     Sequence Label: 
            HCT163_pAAV-eHGT_3Xcore_1137m-minBGprom-ChR2(H134R)-EYFP-WPRE3-bGHpA
FEATURES             Location/Qualifiers
     source          1..6222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      1295..1304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1301..2227
                     /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      2237..2246
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2243..2245,2246..2248,2249..2962)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2288..2309)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2549..2568)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2896..2917
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3049..3069)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3256..3367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3485..3625
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3485..3614
                     /label=AAV2 ITR
     rep_origin      3700..4155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3787..3806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3997..4018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4172..4191)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4291..4313
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4351..4369)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4437..4541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4542..4610,4611..5402)
                     /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(4760..4779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5573..6161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6062..6081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgatttc tttgtctctg tggaagcact taagtgcagg
      181 ctttagttcc aatgacactc aggagcctct ggattccagc actggggatg ggggtggggt
      241 agaacgttct caggcctcac caacccctcc cctgtgtgct gcctttggga gagtcccaag
      301 gcttcagcat tacttaatta attaggcctc tactgctaca taggctcaga ttcaaaagaa
      361 cagagtggcc cacgtcagcc attcccggaa aagtctgatg gctggaagcc agaggactat
      421 gtgtctgcct tgctgccctt ggccagccca tcctgaatgc ccagactcgg acaatggagt
      481 aggtacagaa gggtagattt ctttgtctct gtggaagcac ttaagtgcag gctttagttc
      541 caatgacact caggagcctc tggattccag cactggggat gggggtgggg tagaacgttc
      601 tcaggcctca ccaacccctc ccctgtgtgc tgcctttggg agagtcccaa ggcttcagca
      661 ttacttaatt aattaggcct ctactgctac ataggctcag attcaaaaga acagagtggc
      721 ccacgtcagc cattcccgga aaagtctgat ggctggaagc cagaggacta tgtgtctgcc
      781 ttgctgccct tggccagccc atcctgaatg cccagactcg gacaatggag taggtacaga
      841 agggtagatt tctttgtctc tgtggaagca cttaagtgca ggctttagtt ccaatgacac
      901 tcaggagcct ctggattcca gcactgggga tgggggtggg gtagaacgtt ctcaggcctc
      961 accaacccct cccctgtgtg ctgcctttgg gagagtccca aggcttcagc attacttaat
     1021 taattaggcc tctactgcta cataggctca gattcaaaag aacagagtgg cccacgtcag
     1081 ccattcccgg aaaagtctga tggctggaag ccagaggact atgtgtctgc cttgctgccc
     1141 ttggccagcc catcctgaat gcccagactc ggacaatgga gtaggtacag aagggtacta
     1201 tttcactgag tttgagctcg ggctgggcat aaaagtcagg gcagagccat ctattgctta
     1261 catttgcttc tgggatccag atctttcgaa gctagccacc atggactatg gcggcgcttt
     1321 gtctgccgtc ggacgcgaac ttttgttcgt tactaatcct gtggtggtga acgggtccgt
     1381 cctggtccct gaggatcaat gttactgtgc cggatggatt gaatctcgcg gcacgaacgg
     1441 cgctcagacc gcgtcaaatg tcctgcagtg gcttgcagca ggattcagca ttttgctgct
     1501 gatgttctat gcctaccaaa cctggaaatc tacatgcggc tgggaggaga tctatgtgtg
     1561 cgccattgaa atggttaagg tgattctcga gttctttttt gagtttaaga atccctctat
     1621 gctctacctt gccacaggac accgggtgca gtggctgcgc tatgcagagt ggctgctcac
     1681 ttgtcctgtc atccttatcc gcctgagcaa cctcaccggc ctgagcaacg actacagcag
     1741 gagaaccatg ggactccttg tctcagacat cgggactatc gtgtgggggg ctaccagcgc
     1801 catggcaacc ggctatgtta aagtcatctt cttttgtctt ggattgtgct atggcgcgaa
     1861 cacatttttt cacgccgcca aagcatatat cgagggttat catactgtgc caaagggtcg
     1921 gtgccgccag gtcgtgaccg gcatggcatg gctgtttttc gtgagctggg gtatgttccc
     1981 aattctcttc attttggggc ccgaaggttt tggcgtcctg agcgtctatg gctccaccgt
     2041 aggtcacacg attattgatc tgatgagtaa aaattgttgg gggttgttgg gacactacct
     2101 gcgcgtcctg atccacgagc acatattgat tcacggagat atccgcaaaa ccaccaaact
     2161 gaacatcggc ggaacggaga tcgaggtcga gactctcgtc gaagacgaag ccgaggccgg
     2221 agccgtgcca gcggccgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     2281 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2341 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2401 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc
     2461 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2521 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2581 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2641 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2701 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     2761 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     2821 cgtgctgctg cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa
     2881 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2941 catggacgag ctgtacaagt aatagggcgc gccgcggccg cgaattcgat atcataatca
     3001 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3061 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3121 tttcattttc tcctccttgt ataaatcctg gttagttctt gccacggcgg aactcatcgc
     3181 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggc
     3241 tcgagagatc ttcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3301 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3361 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3421 gcaaggggga ggattgggaa gacaatagca ggcatgagat ctcacgtgcg gaccgagcgg
     3481 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3541 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     3601 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     3661 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     3721 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     3781 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3841 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3901 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3961 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4021 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     4081 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4141 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4201 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4261 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4321 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4381 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4441 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4501 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4561 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4621 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4681 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4741 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4801 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4861 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4921 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4981 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5041 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5101 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5161 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5221 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5281 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5341 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5401 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5461 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5521 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5581 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5641 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5701 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5761 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5821 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5881 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5941 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6001 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6061 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6121 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6181 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
