LOCUS       sequence-9766-ex        7208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian 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 7208)
  AUTHORS   Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, 
            Bamberg E, Nagel G, Gottschalk A, Deisseroth K
  TITLE     Multimodal fast optical interrogation of neural circuitry.
  JOURNAL   Nature. 2007 Apr 5. 446(7136):633-9.
  PUBMED    17410168
REFERENCE   2  (bases 1 to 7208)
  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: "Nature. 
            2007 Apr 5. 446(7136):633-9."
COMMENT     Sequence Label: pcDNA3.1/hChR2(H134R)-EYFP
FEATURES             Location/Qualifiers
     source          1..7208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      957..966
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             963..1889
                     /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      1899..1908
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1905..1907,1908..1910,1911..2624)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1950..1971)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2211..2230)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2558..2579
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2705..2746
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2756..2773
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2796..2813)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2802..3026
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3072..3500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3159..3178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3369..3390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3509..3529)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3514..3844
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3695..3830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3757..3776
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3911..4705
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3965..3984)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4575..4594
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4881..5002
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4918..4937)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4972..4991
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5051..5067)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5051..5067)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5064..5086)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5075..5091
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5099..5105,5106..5123,5124..5129))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5144..5165
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5282..5299)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5453..6041)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5533..5552)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6212..7003,7004..7072))
                     /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     6835..6854
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7073..7177)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattgcc cttgctgcca
      961 ccatggacta tggcggcgct ttgtctgccg tcggacgcga acttttgttc gttactaatc
     1021 ctgtggtggt gaacgggtcc gtcctggtcc ctgaggatca atgttactgt gccggatgga
     1081 ttgaatctcg cggcacgaac ggcgctcaga ccgcgtcaaa tgtcctgcag tggcttgcag
     1141 caggattcag cattttgctg ctgatgttct atgcctacca aacctggaaa tctacatgcg
     1201 gctgggagga gatctatgtg tgcgccattg aaatggttaa ggtgattctc gagttctttt
     1261 ttgagtttaa gaatccctct atgctctacc ttgccacagg acaccgggtg cagtggctgc
     1321 gctatgcaga gtggctgctc acttgtcctg tcatccttat ccgcctgagc aacctcaccg
     1381 gcctgagcaa cgactacagc aggagaacca tgggactcct tgtctcagac atcgggacta
     1441 tcgtgtgggg ggctaccagc gccatggcaa ccggctatgt taaagtcatc ttcttttgtc
     1501 ttggattgtg ctatggcgcg aacacatttt ttcacgccgc caaagcatat atcgagggtt
     1561 atcatactgt gccaaagggt cggtgccgcc aggtcgtgac cggcatggca tggctgtttt
     1621 tcgtgagctg gggtatgttc ccaattctct tcattttggg gcccgaaggt tttggcgtcc
     1681 tgagcgtcta tggctccacc gtaggtcaca cgattattga tctgatgagt aaaaattgtt
     1741 gggggttgtt gggacactac ctgcgcgtcc tgatccacga gcacatattg attcacggag
     1801 atatccgcaa aaccaccaaa ctgaacatcg gcggaacgga gatcgaggtc gagactctcg
     1861 tcgaagacga agccgaggcc ggagccgtgc cagcggccgc caccatggtg agcaagggcg
     1921 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1981 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     2041 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg
     2101 gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     2161 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2221 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2281 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2341 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     2401 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2461 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     2521 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2581 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaacatatg ctctctctaa
     2641 gggcaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg gcccgcggtt
     2701 cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta ccggtcatca
     2761 tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     2821 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2881 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2941 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3001 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3061 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3121 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3181 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg
     3241 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3301 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3361 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3421 ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta
     3481 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3541 ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3601 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3661 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3781 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3841 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     3901 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3961 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4021 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4081 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4141 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4201 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4261 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4321 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4381 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4441 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4501 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4561 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4621 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4681 tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg accgaccaag
     4741 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4801 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4861 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4921 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4981 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     5041 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5101 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5161 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5221 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5281 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5341 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5401 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5461 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5521 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5581 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5641 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5701 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5761 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5821 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5881 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5941 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6001 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6061 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6121 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6181 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6241 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6301 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6361 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6421 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6481 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6541 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6601 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6661 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6721 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6781 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6841 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6901 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6961 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7021 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7081 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7141 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7201 ctgacgtc
//
