LOCUS       sequence-196784-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of EF1a-driven, Cre-dependent, humanized 
            channelrhodopsin E123T/T159C mutant fused to EYFP for optogenetic 
            activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  AUTHORS   Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R,
            Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K
  TITLE     Principles for applying optogenetic tools derived from direct 
            comparative analysis of microbial opsins.
  JOURNAL   Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808.
  PUBMED    22179551
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/nmeth"; 
            journalName: "Nat Methods"; date: "2011-12-18- 18"; volume: "9"; 
            issue: "2"; pages: "159-72"
COMMENT     Sequence Label: pAAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        225..1403
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          456..1394
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1351..1371
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1434..1467)
                     /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(1518..1551)
                     /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(1563..2276,2277..2279,2280..2282))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1608..1629)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1957..1976
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2216..2237
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2279..2288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2298..3224)
                     /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
                     PSMLYLATGHRVQWLRYATWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3221..3230
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3235..3268
                     /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    3319..3352
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3377..3965
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3430..3450)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3848..3859)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3997..4473
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4196..4215)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4513..4653
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4513..4642
                     /label=AAV2 ITR
     rep_origin      4728..5183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4815..4834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5025..5046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5200..5219)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5319..5341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5379..5397)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5465..5569
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5570..5638,5639..6430)
                     /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(5788..5807)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6601..7189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7090..7109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7251..7256,1..124)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc
       61 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
      121 tcctgcggcc gcacgcgtaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc
      181 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
      241 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
      301 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
      361 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
      421 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
      481 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc
      541 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
      601 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
      661 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
      721 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
      781 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
      841 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
      901 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
      961 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     1021 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     1081 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     1141 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt
     1201 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     1261 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     1321 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     1381 ttcttccatt tcaggtgtcg tgaggtaccg gatcctctag agtcgactcc ggaataactt
     1441 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
     1501 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgccc
     1561 tattacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     1621 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg
     1681 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     1741 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     1801 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     1861 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     1921 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     1981 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     2041 tgcttcatgt ggtcggggta gcgggcgaag cactgcaggc cgtagccgaa ggtggtcacg
     2101 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     2161 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     2221 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     2281 atggtggcgg ccgctggcac ggctccggcc tcggcttcgt cttcgacgag agtctcgacc
     2341 tcgatctccg ttccgccgat gttcagtttg gtggttttgc ggatatctcc gtgaatcaat
     2401 atgtgctcgt ggatcaggac gcgcaggtag tgtcccaaca acccccaaca atttttactc
     2461 atcagatcaa taatcgtgtg acctacggtg gagccataga cgctcaggac gccaaaacct
     2521 tcgggcccca aaatgaagag aattgggaac ataccccagc tcacgaaaaa cagccatgcc
     2581 atgccggtca cgacctggcg gcaccgaccc tttggcacag tatgataacc ctcgatatat
     2641 gctttggcgg cgtgaaaaaa tgtgttcgcg ccatagcaca atccaagaca aaagaagatg
     2701 actttaacat agccggttgc catggcgctg gtagcccccc acacgataca cccgatgtct
     2761 gagacaagga gtcccatggt tctcctgctg tagtcgttgc tcaggccggt gaggttgctc
     2821 aggtggataa ggatgacagg acaagtgagc agccaggttg catagcgcag ccactgcacc
     2881 cggtgtcctg tggcaaggta gagcatagag ggattcttaa actcaaaaaa gaactcgaga
     2941 atcaccttaa ccatttcaat ggcgcacaca tagatctcct cccagccgca tgtagatttc
     3001 caggtttggt aggcatagaa catcagcagc aaaatgctga atcctgctgc aagccactgc
     3061 aggacatttg acgcggtctg agcgccgttc gtgccgcgag attcaatcca tccggcacag
     3121 taacattgat cctcagggac caggacggac ccgttcacca ccacaggatt agtaacgaac
     3181 aaaagttcgc gtccgacggc agacaaagcg ccgccatagt ccatggtggc tagcataact
     3241 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc
     3301 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga
     3361 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3421 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3481 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3541 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3601 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     3661 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     3721 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     3781 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta
     3841 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     3901 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     3961 cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc
     4021 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     4081 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     4141 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     4201 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     4261 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     4321 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     4381 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     4441 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga
     4501 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4561 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4621 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     4681 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     4741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4801 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4921 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     4981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5041 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     5101 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5161 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     5221 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5281 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5341 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5401 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5461 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5521 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5581 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5641 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5701 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5761 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5821 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5881 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5941 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6001 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6061 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6121 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6181 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6241 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6301 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6361 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6421 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6481 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6541 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6601 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6661 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6721 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6781 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6841 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6901 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6961 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     7021 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7081 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7141 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7201 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgca
//
