LOCUS       sequence-110864-        7035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to generate AAV virus that will express the 
            ChR2(H134R)-EYFP channelrhodopsin fusion protein in neurons under 
            intersectional control by Dre and Cre recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7035)
  AUTHORS   Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, Li L, van
            der Bourg A, Niino Y, Egolf L, Monetti C, Gu H, Mills M, Cheng A, 
            Tasic B, Nguyen TN, Sunkin SM, Benucci A, Nagy A, Miyawaki A, 
            Helmchen F, Empson RM, Knopfel T, Boyden ES, Reid RC, Carandini M, 
            Zeng H
  TITLE     Transgenic mice for intersectional targeting of neural sensors and 
            effectors with high specificity and performance.
  JOURNAL   Neuron. 2015 Mar 4;85(5):942-58. doi: 10.1016/j.neuron.2015.02.022.
  PUBMED    25741722
REFERENCE   2  (bases 1 to 7035)
  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"; doi: 
            "10.1016/j.neuron.2015.02"; journalName: "Neuron"; date: "2015-03-4-
            4"; volume: "85"; issue: "5"; pages: "942-58"
COMMENT     Sequence Label: AAV phSyn1-RSR-FLEX-ChR2(H134R)-EYFP-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..7035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        177..624
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    637..668
                     /label=rox
                     /bound_moiety="Dre recombinase"
     polyA_signal    714..762
                     /note="synthetic polyadenylation signal"
     polyA_signal    771..1247
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(970..989)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1282..1301)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1326..1374
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1534..1565
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1580..1613)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1713..2423,2424..2426,2427..2429))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1755..1776)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2104..2123
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2363..2384
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2426..2435
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2445..3371)
                     /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"
     CDS             complement(2445..3371)
                     /codon_start=1
                     /product="N-terminal fragment of channelrhodopsin-2 from 
                     the alga Chlamydomonas reinhardtii (Nagel et al., 2003)"
                     /label=ChR2
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3368..3377
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3409..3442
                     /label=loxJTZ17
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3461..4049
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3514..3534)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3932..3943)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4067..4084)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4073..4280
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4298..4438
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4298..4427
                     /label=AAV2 ITR
     rep_origin      4513..4968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4600..4619)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4810..4831
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4985..5004)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5104..5126
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5164..5182)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5250..5354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5355..5423,5424..6215)
                     /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(5573..5592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6386..6974
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6875..6894
                     /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 tgcggcccga acgcgtctgc agagggcctg cgtatgagtg
      181 caagtgggtt ttaggaccag gatgaggcgg ggtgggggtg cctacctgac gaccgacccc
      241 gacccactgg acaagcaccc aacccccatt ccccaaattg cgcatcccct atcagagagg
      301 gggaggggaa acaggatgcg gcgaggcgcg tgcgcactgc cagcttcagc accgcggaca
      361 gtgccttcgc ccccgcctgg cggcgcgcgc caccgccgcc tcagcactga aggcgcgctg
      421 acgtcactcg ccggtccccc gcaaactccc cttcccggcc accttggtcg cgtccgcgcc
      481 gccgccggcc cagccggacc gcaccacgcg aggcgcgaga taggggggca cgggcgcgac
      541 catctgcgct gcggcgccgg cgactcagcg ctgcctcagt ctgcggtggg cagcggagga
      601 gtcgtgtcgt gcctgagagc gcagacgcgt ttcgaataac tttaaataat tggcattatt
      661 taaagttacc tcagcaccat ggctagcggc agcctcggag tttgaataga tagaataaaa
      721 tatctttatt ttcattccat ctgtgtgttg gttttttgtg tgagatctac gggtggcatc
      781 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
      841 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
      901 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
      961 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     1021 tctccgcctc ctgggttcaa gcgattctcc tgcctcaggc tcccgagttg ttgggattcc
     1081 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     1141 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     1201 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg cctcagctaa
     1261 ttgagtaggg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     1321 tatgacggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcataaac
     1381 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     1441 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     1501 ggctcgcagc caacgtcggg gcggcaggcc gtgtaacttt aaataattgg cattatttaa
     1561 agttactcga ggttaacttt accgttcgta tagcatacat tatacgaagt tatctgcgtc
     1621 gaccttgttt aaactccgga gctagcaccg gtggcgcgcc atttaaatgg ccggcctgaa
     1681 ttcacttgcc ggcggccgct ttatccggaa cccttgtaca gctcgtccat gccgagagtg
     1741 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     1801 ttgctcaggg cggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     1861 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     1921 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     1981 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     2041 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     2101 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     2161 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac
     2221 tgcaggccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     2281 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     2341 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     2401 gtgaacagct cctcgccctt gctcaccatg gtggcggccg cgggtactgc gccagcctcg
     2461 gcctcgtcct ccaccagcgt ctcgacctca atctcagtgc caccaatgtt caatttggtg
     2521 gtcttgcgaa tgtcgccgtg gatgaggata tgctcgtgga tcagcacgcg caggtagtgg
     2581 ccgagcagac cccagcagtt cttcgacatc aggtcaatga tggtgtggcc gacggtggag
     2641 ccgtacacgc tcaggacgcc gaagccctcg gggccgagga tgaacaggat ggggaacata
     2701 ccccatgata cgaagaagag ccaagccatg ccagtcacca cctggcgaca ccggcccttc
     2761 ggcacggtgt ggtaaccctc gatgtaggcc ttggcagcgt gaaagaacgt gttagcacca
     2821 taacacagac ccaggcagaa gaagatgacc ttgacgtatc cggtggccat ggcggaagtg
     2881 gcgccccaca caattgtgcc aatatcagac acaagcagac ccatggtgcg cctgctgtag
     2941 tcgttggaca agcccgtcag gtttgacagg cgaatgagaa tgaccgggca ggtgagaagc
     3001 cactcggcgt aacgcaacca ctggacgcgg tggcctgtgg ctagatacag catggacggg
     3061 ttcttaaact cgaagaagaa ctcgagaatc accttgacca tctcgatagc gcacacatag
     3121 atctcctccc agccgcaggt tgacttccat gtttggtagg cgtaaaacat aagcagtagg
     3181 atggagaagc cagcagcaag ccattgcagc acgttcgacg ccgtttgggc accgtttgtg
     3241 ccacgcgact caatccagcc cgcgcagtaa cactggtcct caggcacaag tacagagcca
     3301 ttgacgacta ctgggttcgt tacaaatagc agctcgcgcc caacggcact cagggcgcct
     3361 ccataatcca tggtggcaag gccggccagg atttaaatgg cgcgccaaat aaattgctat
     3421 aatgtatgct atacgaagtt atcactagtc gcttatcgat aatcaacctc tggattacaa
     3481 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3541 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3601 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3661 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3721 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3781 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3841 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     3901 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3961 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4021 tcggatctcc ctttgggccg cctccccgca tcgataccgc ctagggcctc gactgtgcct
     4081 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     4141 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     4201 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     4261 aatagcaggc atgctgggga ttaattaatt gggccgcagg aacccctagt gatggagttg
     4321 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4381 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4441 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4501 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4561 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4621 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4681 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     4741 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4801 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     4861 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4921 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     4981 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5041 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5101 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5161 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5221 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5281 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5341 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5401 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5461 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5521 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5581 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5641 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5701 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5761 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5821 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5881 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5941 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6001 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6061 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6121 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6181 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6241 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6301 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6361 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6421 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6481 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6541 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6601 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6661 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6721 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6781 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6841 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6901 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6961 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7021 cttttgctca catgt
//
