LOCUS       sequence-466768-        6382 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 6382)
  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 6382)
  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: 
            HCT164_pAAV-3X-eHGT_1139m-minBGprom-ChR2(H134R)-EYFP-WPRE3-bGHpA
FEATURES             Location/Qualifiers
     source          1..6382
                     /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      1455..1464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1461..2387
                     /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      2397..2406
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2403..2405,2406..2408,2409..3122)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2448..2469)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2709..2728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3056..3077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3209..3229)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3416..3527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3645..3785
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3645..3774
                     /label=AAV2 ITR
     rep_origin      3860..4315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3947..3966)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4157..4178
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4332..4351)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4451..4473
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4511..4529)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4597..4701
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4702..4770,4771..5562)
                     /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(4920..4939)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5733..6321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6222..6241
                     /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 gcgtaaatat tacaatacta aaaaaggaaa gacaagaaat
      181 gaagccagta gatatatttt caatagcata gtatatttcc agttaccaca tttttttgct
      241 gatgtttgta cttaaatgta tattaaaaag tttcttatcc caagtaaaga gcattactaa
      301 aattactgtg tgccactctg aaagatgaga gaaacccata ttcagtttcc ataatgaaat
      361 ccatatttgc ttggttgtga tccaaattgt ttttgaaaag tatatattta attgaggcag
      421 atgaagtggt ccaaaattta ataagtatta aaaaaaagga aaatttaaag agaaaatttc
      481 caattaaaga tgagtacctg atgattgctc aggcccaatt attaatattg attaatgttt
      541 tgtactttaa atattacaat actaaaaaag gaaagacaag aaatgaagcc agtagatata
      601 ttttcaatag catagtatat ttccagttac cacatttttt tgctgatgtt tgtacttaaa
      661 tgtatattaa aaagtttctt atcccaagta aagagcatta ctaaaattac tgtgtgccac
      721 tctgaaagat gagagaaacc catattcagt ttccataatg aaatccatat ttgcttggtt
      781 gtgatccaaa ttgtttttga aaagtatata tttaattgag gcagatgaag tggtccaaaa
      841 tttaataagt attaaaaaaa aggaaaattt aaagagaaaa tttccaatta aagatgagta
      901 cctgatgatt gctcaggccc aattattaat attgattaat gttttgtact ttaaatatta
      961 caatactaaa aaaggaaaga caagaaatga agccagtaga tatattttca atagcatagt
     1021 atatttccag ttaccacatt tttttgctga tgtttgtact taaatgtata ttaaaaagtt
     1081 tcttatccca agtaaagagc attactaaaa ttactgtgtg ccactctgaa agatgagaga
     1141 aacccatatt cagtttccat aatgaaatcc atatttgctt ggttgtgatc caaattgttt
     1201 ttgaaaagta tatatttaat tgaggcagat gaagtggtcc aaaatttaat aagtattaaa
     1261 aaaaaggaaa atttaaagag aaaatttcca attaaagatg agtacctgat gattgctcag
     1321 gcccaattat taatattgat taatgttttg tactttacta tttcactgag tttgagctcg
     1381 ggctgggcat aaaagtcagg gcagagccat ctattgctta catttgcttc tgggatccag
     1441 atctttcgaa gctagccacc atggactatg gcggcgcttt gtctgccgtc ggacgcgaac
     1501 ttttgttcgt tactaatcct gtggtggtga acgggtccgt cctggtccct gaggatcaat
     1561 gttactgtgc cggatggatt gaatctcgcg gcacgaacgg cgctcagacc gcgtcaaatg
     1621 tcctgcagtg gcttgcagca ggattcagca ttttgctgct gatgttctat gcctaccaaa
     1681 cctggaaatc tacatgcggc tgggaggaga tctatgtgtg cgccattgaa atggttaagg
     1741 tgattctcga gttctttttt gagtttaaga atccctctat gctctacctt gccacaggac
     1801 accgggtgca gtggctgcgc tatgcagagt ggctgctcac ttgtcctgtc atccttatcc
     1861 gcctgagcaa cctcaccggc ctgagcaacg actacagcag gagaaccatg ggactccttg
     1921 tctcagacat cgggactatc gtgtgggggg ctaccagcgc catggcaacc ggctatgtta
     1981 aagtcatctt cttttgtctt ggattgtgct atggcgcgaa cacatttttt cacgccgcca
     2041 aagcatatat cgagggttat catactgtgc caaagggtcg gtgccgccag gtcgtgaccg
     2101 gcatggcatg gctgtttttc gtgagctggg gtatgttccc aattctcttc attttggggc
     2161 ccgaaggttt tggcgtcctg agcgtctatg gctccaccgt aggtcacacg attattgatc
     2221 tgatgagtaa aaattgttgg gggttgttgg gacactacct gcgcgtcctg atccacgagc
     2281 acatattgat tcacggagat atccgcaaaa ccaccaaact gaacatcggc ggaacggaga
     2341 tcgaggtcga gactctcgtc gaagacgaag ccgaggccgg agccgtgcca gcggccgcca
     2401 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2461 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2521 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2581 ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
     2641 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2701 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2761 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2821 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2881 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2941 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3001 actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     3061 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     3121 aatagggcgc gccgcggccg cgaattcgat atcataatca acctctggat tacaaaattt
     3181 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3241 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3301 ataaatcctg gttagttctt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3361 ggacaggggc tcggctgttg ggcactgaca attccgtggc tcgagagatc ttcgactgtg
     3421 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3481 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3541 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3601 gacaatagca ggcatgagat ctcacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     3661 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3721 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     3781 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3841 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3901 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     3961 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4021 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4081 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4141 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     4201 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4261 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4321 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4381 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4441 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4501 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     4561 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4621 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4681 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4741 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4801 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     4861 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     4921 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     4981 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5041 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5101 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5161 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5221 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5281 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5341 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5401 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5461 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5521 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5581 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5641 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5701 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5761 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5821 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5881 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5941 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6001 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6061 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6121 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6181 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6241 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6301 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6361 tgctggcctt ttgctcacat gt
//
