LOCUS       sequence-246739-        7108 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent AAV expression of humanized ChR2 (with H134R mutation)
            fused to eYFP for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7108)
  AUTHORS   Rajendran PS, Challis RC, Fowlkes CC, Hanna P, Tompkins JD, Jordan 
            MC, Hiyari S, Gabris-Weber BA, Greenbaum A, Chan KY, Deverman BE, 
            Munzberg H, Ardell JL, Salama G, Gradinaru V, Shivkumar K
  TITLE     Identification of peripheral neural circuits that regulate heart 
            rate using optogenetic and viral vector strategies.
  JOURNAL   Nat Commun. 2019 Apr 26;10(1):1944. doi: 10.1038/s41467-019-09770-1.
  PUBMED    31028266
REFERENCE   2  (bases 1 to 7108)
  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: "Nat 
            Commun."; date: "2019-04-26"; volume: "10(1):1944. doi"; pages: " 
            10.1038/s41467-019-09770-1"
COMMENT     Sequence Label: pAAV-CAG-DIO-ChR2(H134R)-eYFP
FEATURES             Location/Qualifiers
     source          1..7108
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..792
                     /label=chicken beta-actin promoter
     primer_bind     1122..1144
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1206..1225
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(1292..1325)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1376..1409)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1421..2134,2135..2137,2138..2140))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1466..1487)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1815..1834
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2074..2095
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2137..2146
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2156..3082)
                     /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      3079..3088
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3093..3126
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3177..3210
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3235..3823
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3288..3308)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3706..3717)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3855..4331
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4054..4073)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4371..4511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4371..4500
                     /label=AAV2 ITR
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5177..5199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5237..5255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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(5646..5665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6459..7047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6948..6967
                     /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 gcgtaacact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      661 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      781 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      841 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      901 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      961 tggctgcgtg aaagccttga ggggctccgg gagcgccggc aggaaggaaa tgggcgggga
     1021 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1081 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1141 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1201 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgcggcccaa
     1261 cggtaccgga tcctctagag tcgactccgg aataacttcg tataggatac tttatacgaa
     1321 gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc tcttaataac
     1381 ttcgtatagc atacattata cgaagttatg gcgcgcccta ttacttgtac agctcgtcca
     1441 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     1501 cgttggggtc tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca
     1561 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
     1621 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca
     1681 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
     1741 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
     1801 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
     1861 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
     1921 gggcgaagca ctgcaggccg tagccgaagg tggtcacgag ggtgggccag ggcacgggca
     1981 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
     2041 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
     2101 gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcggcc gctggcacgg
     2161 ctccggcctc ggcttcgtct tcgacgagag tctcgacctc gatctccgtt ccgccgatgt
     2221 tcagtttggt ggttttgcgg atatctccgt gaatcaatat gtgctcgtgg atcaggacgc
     2281 gcaggtagtg tcccaacaac ccccaacaat ttttactcat cagatcaata atcgtgtgac
     2341 ctacggtgga gccatagacg ctcaggacgc caaaaccttc gggccccaaa atgaagagaa
     2401 ttgggaacat accccagctc acgaaaaaca gccatgccat gccggtcacg acctggcggc
     2461 accgaccctt tggcacagta tgataaccct cgatatatgc tttggcggcg tgaaaaaatg
     2521 tgttcgcgcc atagcacaat ccaagacaaa agaagatgac tttaacatag ccggttgcca
     2581 tggcgctggt agccccccac acgatagtcc cgatgtctga gacaaggagt cccatggttc
     2641 tcctgctgta gtcgttgctc aggccggtga ggttgctcag gcggataagg atgacaggac
     2701 aagtgagcag ccactctgca tagcgcagcc actgcacccg gtgtcctgtg gcaaggtaga
     2761 gcatagaggg attcttaaac tcaaaaaaga actcgagaat caccttaacc atttcaatgg
     2821 cgcacacata gatctcctcc cagccgcatg tagatttcca ggtttggtag gcatagaaca
     2881 tcagcagcaa aatgctgaat cctgctgcaa gccactgcag gacatttgac gcggtctgag
     2941 cgccgttcgt gccgcgagat tcaatccatc cggcacagta acattgatcc tcagggacca
     3001 ggacggaccc gttcaccacc acaggattag taacgaacaa aagttcgcgt ccgacggcag
     3061 acaaagcgcc gccatagtcc atggtggcta gcataacttc gtataaagta tcctatacga
     3121 agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa
     3181 cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat cgataatcaa
     3241 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3301 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3361 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3421 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3481 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3541 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3601 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat
     3661 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3721 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     3781 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg
     3841 ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     3901 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     3961 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     4021 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     4081 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     4141 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     4201 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     4261 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     4321 tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct
     4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4501 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7081 gccttttgct ggccttttgc tcacatgt
//
