LOCUS       sequence-196781-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of humanized ChR2 with E123T/H134R mutations fused to
            EYFP driven by EF1a promoter for optogenetic activation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  AUTHORS   Gunaydin LA, Yizhar O, Berndt A, Sohal VS, Deisseroth K, Hegemann P
  TITLE     Ultrafast optogenetic control.
  JOURNAL   Nat Neurosci. 2010 Mar;13(3):387-92. doi: 10.1038/nn.2495.
  PUBMED    20081849
REFERENCE   2  (bases 1 to 7256)
  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: "10.1038/nn"; 
            journalName: "Nat Neurosci"; date: "2010-03"; volume: "13"; issue: 
            "3"; pages: "387-92"
COMMENT     Sequence Label: pAAV-Ef1a-DIO ChETA-EYFP
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   51..180
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        281..1459
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          512..1450
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1407..1427
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1490..1523)
                     /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(1574..1607)
                     /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(1619..2332,2333..2335,2336..2338))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1664..1685)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2013..2032
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2272..2293
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2335..2344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2354..3280)
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3277..3286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3291..3324
                     /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    3375..3408
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3433..4021
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3486..3506)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3904..3915)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4053..4529
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4252..4271)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4569..4709
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4569..4698
                     /label=AAV2 ITR
     rep_origin      4784..5239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4871..4890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5081..5102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5256..5275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5375..5397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5435..5453)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5521..5625
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5626..5694,5695..6486)
                     /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(5844..5863)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6657..7245
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7146..7165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
       61 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      181 gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga ggaatctttg
      241 cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg
      301 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      361 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      421 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
      481 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
      541 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
      601 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
      661 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
      721 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
      781 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
      841 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
      901 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
      961 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1021 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1081 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     1141 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1201 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1261 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1321 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1381 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     1441 tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa taacttcgta
     1501 taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
     1561 atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc gcgccctatt
     1621 acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga
     1681 ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactggtag ctcaggtagt
     1741 ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg
     1801 cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc ttgatgccgt
     1861 tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac tccagcttgt
     1921 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     1981 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     2041 agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     2101 tcatgtggtc ggggtagcgg gcgaagcact gcaggccgta gccgaaggtg gtcacgaggg
     2161 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     2221 aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     2281 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgg
     2341 tggcggccgc tggcacggct ccggcctcgg cttcgtcttc gacgagagtc tcgacctcga
     2401 tctccgttcc gccgatgttc agtttggtgg ttttgcggat atctccgtga atcaatatgt
     2461 gctcgtggat caggacgcgc aggtagtgtc ccaacaaccc ccaacaattt ttactcatca
     2521 gatcaataat cgtgtgacct acggtggagc catagacgct caggacgcca aaaccttcgg
     2581 gccccaaaat gaagagaatt gggaacatac cccagctcac gaaaaacagc catgccatgc
     2641 cggtcacgac ctggcggcac cgaccctttg gcacagtatg ataaccctcg atatatgctt
     2701 tggcggcgtg aaaaaatgtg ttcgcgccat agcacaatcc aagacaaaag aagatgactt
     2761 taacatagcc ggttgccatg gcgctggtag ccccccacac gatagtcccg atgtctgaga
     2821 caaggagtcc catggttctc ctgctgtagt cgttgctcag gccggtgagg ttgctcaggc
     2881 ggataaggat gacaggacaa gtgagcagcc aggttgcata gcgcagccac tgcacccggt
     2941 gtcctgtggc aaggtagagc atagagggat tcttaaactc aaaaaagaac tcgagaatca
     3001 ccttaaccat ttcaatggcg cacacataga tctcctccca gccgcatgta gatttccagg
     3061 tttggtaggc atagaacatc agcagcaaaa tgctgaatcc tgctgcaagc cactgcagga
     3121 catttgacgc ggtctgagcg ccgttcgtgc cgcgagattc aatccatccg gcacagtaac
     3181 attgatcctc agggaccagg acggacccgt tcaccaccac aggattagta acgaacaaaa
     3241 gttcgcgtcc gacggcagac aaagcgccgc catagtccat ggtggctagc ataacttcgt
     3301 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     3361 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc
     3421 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3481 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3541 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3601 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3661 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3721 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3781 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3841 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3901 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3961 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     4021 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4081 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4141 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4201 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     4261 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     4321 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     4381 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     4441 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     4501 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     4561 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4621 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4681 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4741 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4801 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4861 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4921 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4981 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5041 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5101 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5161 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5221 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5281 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5341 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5401 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5461 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5521 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5581 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5641 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5701 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5761 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5821 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5881 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5941 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6001 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6061 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6121 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6181 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6241 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6301 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6361 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6421 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6481 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6541 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6601 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6661 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6721 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6781 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6841 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6901 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6961 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7021 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7081 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7141 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7201 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaa
//
