LOCUS       sequence-436383-        7028 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 Flp and Cre recombinases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7028)
  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 7028)
  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-FSF-FLEX-ChR2(H134R)-EYFP-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..7028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   43..183
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   54..183
                     /label=AAV2 ITR
     promoter        219..666
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    679..712
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    758..806
                     /note="synthetic polyadenylation signal"
     polyA_signal    815..1291
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1014..1033)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1326..1345)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1370..1418
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1578..1611
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(1626..1659)
                     /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(1759..2469,2470..2472,2473..2475))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1801..1822)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2150..2169
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2409..2430
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2472..2481
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2491..3417)
                     /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(2491..3417)
                     /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      3414..3423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3455..3488
                     /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    3507..4095
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3560..3580)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3978..3989)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4113..4130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4119..4326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4344..4473
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4535..5123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4615..4634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5294..6085,6086..6154))
                     /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     5917..5936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6155..6259)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6327..6345
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6383..6405)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6505..6524
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6541..6996)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6678..6699)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6890..6909
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg
       61 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
      121 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
      181 cctgcggccc gaacgcgtct gcagagggcc tgcgtatgag tgcaagtggg ttttaggacc
      241 aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac
      301 ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg
      361 cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct
      421 ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc
      481 ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga
      541 ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc
      601 ggcgactcag cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga
      661 gcgcagacgc gtttcgaaga agttcctatt ctctagaaag tataggaact tccctcagca
      721 ccatggctag cggcagcctc ggagtttgaa tagatagaat aaaatatctt tattttcatt
      781 ccatctgtgt gttggttttt tgtgtgagat ctacgggtgg catccctgtg acccctcccc
      841 agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa
      901 attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg
      961 ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg
     1021 ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt
     1081 tcaagcgatt ctcctgcctc aggctcccga gttgttggga ttccaggcat gcatgaccag
     1141 gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt
     1201 ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca
     1261 ggcgtgaacc actgctccct tccctgtcct tctgcctcag ctaattgagt aggggggagg
     1321 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     1381 agacagaata aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     1441 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     1501 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     1561 cggggcggca ggccgtggaa gttcctattc tctagaaagt ataggaactt cctcgaggtt
     1621 aactttaccg ttcgtatagc atacattata cgaagttatc tgcgtcgacc ttgtttaaac
     1681 tccggagcta gcaccggtgg cgcgccattt aaatggccgg cctgaattca cttgccggcg
     1741 gccgctttat ccggaaccct tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     1801 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     1861 ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     1921 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     1981 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     2041 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     2101 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     2161 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     2221 gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc
     2281 gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     2341 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     2401 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2461 gcccttgctc accatggtgg cggccgcggg tactgcgcca gcctcggcct cgtcctccac
     2521 cagcgtctcg acctcaatct cagtgccacc aatgttcaat ttggtggtct tgcgaatgtc
     2581 gccgtggatg aggatatgct cgtggatcag cacgcgcagg tagtggccga gcagacccca
     2641 gcagttcttc gacatcaggt caatgatggt gtggccgacg gtggagccgt acacgctcag
     2701 gacgccgaag ccctcggggc cgaggatgaa caggatgggg aacatacccc atgatacgaa
     2761 gaagagccaa gccatgccag tcaccacctg gcgacaccgg cccttcggca cggtgtggta
     2821 accctcgatg taggccttgg cagcgtgaaa gaacgtgtta gcaccataac acagacccag
     2881 gcagaagaag atgaccttga cgtatccggt ggccatggcg gaagtggcgc cccacacaat
     2941 tgtgccaata tcagacacaa gcagacccat ggtgcgcctg ctgtagtcgt tggacaagcc
     3001 cgtcaggttt gacaggcgaa tgagaatgac cgggcaggtg agaagccact cggcgtaacg
     3061 caaccactgg acgcggtggc ctgtggctag atacagcatg gacgggttct taaactcgaa
     3121 gaagaactcg agaatcacct tgaccatctc gatagcgcac acatagatct cctcccagcc
     3181 gcaggttgac ttccatgttt ggtaggcgta aaacataagc agtaggatgg agaagccagc
     3241 agcaagccat tgcagcacgt tcgacgccgt ttgggcaccg tttgtgccac gcgactcaat
     3301 ccagcccgcg cagtaacact ggtcctcagg cacaagtaca gagccattga cgactactgg
     3361 gttcgttaca aatagcagct cgcgcccaac ggcactcagg gcgcctccat aatccatggt
     3421 ggcaaggccg gccaggattt aaatggcgcg ccaaataaat tgctataatg tatgctatac
     3481 gaagttatca ctagtcgctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     3541 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3601 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3661 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3721 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3781 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3841 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3901 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3961 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     4021 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4081 gggccgcctc cccgcatcga taccgcctag ggcctcgact gtgccttcta gttgccagcc
     4141 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4201 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4261 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4321 tggggattaa ttaattgggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     4381 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct
     4441 cagtgagcga gcgagcgcgc agctgcctgc aggacatgtg agcaaaaggc cagcaaaagg
     4501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5161 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5221 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5281 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5341 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5401 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5461 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5521 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5581 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5641 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5701 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5761 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5821 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5881 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5941 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6001 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6061 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6121 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6181 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6241 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     6301 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6361 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6421 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6481 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     6541 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6601 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga
     6661 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     6721 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca
     6781 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     6841 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     6901 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6961 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg
     7021 gtgtgaaa
//
