LOCUS       sequence-263449-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ChR2-GFP and Halo-YFP for bi-directional control of 
            neuronal activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  AUTHORS   Han X, Qian X, Stern P, Chuong AS, Boyden ES
  TITLE     Informational lesions: optical perturbation of spike timing and 
            neural synchrony via microbial opsin gene fusions.
  JOURNAL   Front Mol Neurosci. 2009 Aug 27:2:12. doi: 10.3389/neuro.02.012.2009
  PUBMED    19753326
REFERENCE   2  (bases 1 to 7369)
  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: "Front Mol 
            Neurosci. 2009 Aug 27:2:12. doi: 10.3389/neuro.02.012.2009"
COMMENT     Sequence Label: ChR2-2A-Halo
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      602..611
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             617..1540
                     /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="DYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTN
                     GAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNP
                     SMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGA
                     TSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSW
                     GMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIR
                     KTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             617..1540
                     /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="DYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTN
                     GAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNP
                     SMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGA
                     TSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSW
                     GMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIR
                     KTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             join(1562..1564,1565..2275)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1562..1564,1565..2275)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2297..2362
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(3254..3256,3257..3970)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNRHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     polyA_signal    4229..4350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4266..4285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4320..4339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4357..4812)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4494..4515)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4706..4725
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4839..4943
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4968..4988)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5012..5222
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5034..5230
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5081..5216
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5265..6059
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5265..6059
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5319..5338)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5929..5948
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6247..6266)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6291..6338
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6667..7255
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7156..7175
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagaccgg
      601 tgccaccatg ggtaccgact atggcggcgc tttgtctgcc gtcggacgcg aacttttgtt
      661 cgttactaat cctgtggtgg tgaacgggtc cgtcctggtc cctgaggatc aatgttactg
      721 tgccggatgg attgaatctc gcggcacgaa cggcgctcag accgcgtcaa atgtcctgca
      781 gtggcttgca gcaggattca gcattttgct gctgatgttc tatgcctacc aaacctggaa
      841 atctacatgc ggctgggagg agatctatgt gtgcgccatt gaaatggtta aggtgattct
      901 cgagttcttt tttgagttta agaatccctc tatgctctac cttgccacag gacaccgggt
      961 gcagtggctg cgctatgcag agtggctgct cacttgtcct gtcatcctta tccacctgag
     1021 caacctcacc ggcctgagca acgactacag caggagaacc atgggactcc ttgtctcaga
     1081 catcgggact atcgtgtggg gggctaccag cgccatggca accggctatg ttaaagtcat
     1141 cttcttttgt cttggattgt gctatggcgc gaacacattt tttcacgccg ccaaagcata
     1201 tatcgagggt tatcatactg tgccaaaggg tcggtgccgc caggtcgtga ccggcatggc
     1261 atggctgttt ttcgtgagct ggggtatgtt cccaattctc ttcattttgg ggcccgaagg
     1321 ttttggcgtc ctgagcgtct atggctccac cgtaggtcac acgattattg atctgatgag
     1381 taaaaattgt tgggggttgt tgggacacta cctgcgcgtc ctgatccacg agcacatatt
     1441 gattcacgga gatatccgca aaaccaccaa actgaacatc ggcggaacgg agatcgaggt
     1501 cgagactctc gtcgaagacg aagccgaggc cggagccgtg ccagcggcac cggtagtagc
     1561 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1621 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1681 caagctgacc ctgaagttca tttgcaccac cggcaagctg cccgtgccct ggcccaccct
     1741 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1801 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1861 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1921 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1981 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2041 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2101 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2161 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2221 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagaaaca
     2281 gaaaattgtg gcaccagtga aacagacttt gaattttgac cttctcaagt tggcgggaga
     2341 cgtcgagtcc aaccctgggc cctctagaac tgagaccctc ccacccgtga ctgaaagcgc
     2401 cgtcgctctg caagcagagg ttacccagcg ggagctgttc gagttcgtcc tcaacgaccc
     2461 cctcctggct tctagcctct acatcaacat tgctctggca ggcctgtcta tactgctgtt
     2521 cgtcttcatg accaggggac tcgatgaccc tagggctaaa ctgattgcag tgagcacaat
     2581 tctggttccc gtggtctcta tcgcttccta cactgggctg gcatctggtc tcacaatcag
     2641 tgtcctggaa atgccagctg gccactttgc cgaagggagt tctgtcatgc tgggaggcga
     2701 agaggtcgat ggggttgtca caatgtgggg tcgctacctc acctgggctc tcagtacccc
     2761 catgatcctg ctggcactcg gactcctggc cggaagtaac gccaccaaac tcttcactgc
     2821 tattacattc gatatcgcca tgtgcgtgac cgggctcgca gctgccctca ccaccagcag
     2881 ccatctgatg agatggtttt ggtatgccat ctcttgtgcc tgctttctgg tggtgctgta
     2941 tatcctgctg gtggagtggg ctcaggatgc caaggctgca gggacagccg acatgtttaa
     3001 tacactgaag ctgctcactg tggtgatgtg gctgggttac cctatcgttt gggcactcgg
     3061 cgtggaggga atcgcagttc tgcctgttgg tgtgacaagc tggggctact ccttcctgga
     3121 cattgtggcc aagtatattt ttgcctttct gctgctgaat tatctgactt ccaatgagtc
     3181 cgtggtgtcc ggctccatac tggacgtgcc atccgccagc ggcacacctg ccgatgaccg
     3241 accggtagta gcagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     3301 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     3361 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     3421 ctggcccacc ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga
     3481 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     3541 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     3601 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     3661 cctggggcac aagctggagt acaactacaa cagacacaac gtctatatca tggccgacaa
     3721 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3781 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3841 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     3901 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3961 gtacaagtaa gaattccggc tagaagccag gagggggtcg ttgaggacga actcgaacag
     4021 ctcccgctgg gtaacctctg cttgcagagc gacggcgctt tcagtcacgg gtgggagggt
     4081 ctcagttcta gagaattcac atgttgagcg gccgcgactc tagatcataa tcagccatac
     4141 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     4201 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     4261 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4321 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     4381 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4441 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     4501 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     4561 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     4621 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     4681 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     4741 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     4801 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4861 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4921 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     4981 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     5041 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     5101 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     5161 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     5221 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     5281 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     5341 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     5401 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     5461 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     5521 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     5581 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     5641 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     5701 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     5761 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     5821 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     5881 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5941 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     6001 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     6061 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     6121 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     6181 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     6241 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     6301 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     6361 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     6421 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     6481 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     6541 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6601 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6661 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6721 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6781 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6841 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6901 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6961 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7021 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7081 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7141 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7201 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7261 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7321 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
