LOCUS       sequence-196782-        7255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Ef1a-driven, cre-dependent, stabilized step 
            function opsin (SSFO) for optogenetic activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7255)
  AUTHORS   Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O'Shea DJ, 
            Sohal VS, Goshen I, Finkelstein J, Paz JT, Stehfest K, Fudim R, 
            Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K
  TITLE     Neocortical excitation/inhibition balance in information processing 
            and social dysfunction.
  JOURNAL   Nature. 2011 Jul 27;477(7363):171-8. doi: 10.1038/nature10360.
  PUBMED    21796121
REFERENCE   2  (bases 1 to 7255)
  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"; journalName: 
            "Nature"; date: "2011-07-27- 27"; volume: "477"; issue: "7363"; 
            pages: "171-8"
COMMENT     Sequence Label: pAAV-Ef1a-DIO hChR2(C128S/D156A)-EYFP
FEATURES             Location/Qualifiers
     source          1..7255
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   36..165
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        266..1444
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          497..1435
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1392..1412
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1475..1508)
                     /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(1559..1592)
                     /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(1601..2314,2315..2317,2318..2320))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1646..1667)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1995..2014
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2254..2275
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2317..2326
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2336..3262)
                     /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
                     PSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTMGLLVSAIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3259..3268
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3275..3308
                     /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    3359..3392
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3417..4005
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3470..3490)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3888..3899)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4037..4513
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4236..4255)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4553..4693
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4553..4682
                     /label=AAV2 ITR
     rep_origin      4768..5223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4855..4874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5065..5086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5240..5259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5359..5381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5419..5437)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5505..5609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5610..5678,5679..6470)
                     /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(5828..5847)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6641..7229
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7130..7149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcctggcc ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc
       61 gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
      121 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgta
      181 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
      241 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
      301 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
      361 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
      421 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
      481 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
      541 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
      601 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
      661 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
      721 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
      781 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
      841 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
      901 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
      961 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     1021 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     1081 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     1141 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1201 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1261 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     1321 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     1381 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     1441 gtgaggtacc ggatcctcta gagtcgactc cggaataact tcgtatagga tactttatac
     1501 gaagttatgc agaatggtag ctggattgta gctgctatta gcaatatgaa acctcttaat
     1561 aacttcgtat agcatacatt atacgaagtt atggcgcgcc ttacttgtac agctcgtcca
     1621 tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct
     1681 cgttggggtc tttgctcagg gcggactggt agctcaggta gtggttgtcg ggcagcagca
     1741 cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt
     1801 cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca
     1861 tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt
     1921 cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact
     1981 tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga
     2041 cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc
     2101 gggcgaagca ctgcaggccg tagccgaagg tggtcacgag ggtgggccag ggcacgggca
     2161 gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc
     2221 cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg
     2281 gcaccacccc ggtgaacagc tcctcgccct tgctcaccat ggtggcggcc gctggcacgg
     2341 ctccggcctc ggcttcgtct tcgacgagag tctcgacctc gatctccgtt ccgccgatgt
     2401 tcagtttggt ggttttgcgg atatctccgt gaatcaatat gtgctcgtgg atcaggacgc
     2461 gcaggtagtg tcccaacaac ccccaacaat ttttactcat cagatcaata atcgtgtgac
     2521 ctacggtgga gccatagacg ctcaggacgc caaaaccttc gggccccaaa atgaagagaa
     2581 ttgggaacat accccagctc acgaaaaaca gccatgccat gccggtcacg acctggcggc
     2641 accgaccctt tggcacagta tgataaccct cgatatatgc tttggcggcg tgaaaaaatg
     2701 tgttcgcgcc atagcacaat ccaagacaaa agaagatgac tttaacatag ccggttgcca
     2761 tggcgctggt agccccccac acgatagtcc cgatggctga gacaaggagt cccatggttc
     2821 tcctgctgta gtcgttgctc aggccggtga ggttgctcag gtggataagg atgacaggag
     2881 aagtgagcag ccactctgca tagcgcagcc actgcacccg gtgtcctgtg gcaaggtaga
     2941 gcatagaggg attcttaaac tcaaaaaaga actcgagaat caccttaacc atttcaatgg
     3001 cgcacacata gatctcctcc cagccgcatg tagatttcca ggtttggtag gcatagaaca
     3061 tcagcagcaa aatgctgaat cctgctgcaa gccactgcag gacatttgac gcggtctgag
     3121 cgccgttcgt gccgcgagat tcaatccatc cggcacagta acattgatcc tcagggacca
     3181 ggacggaccc gttcaccacc acaggattag taacgaacaa aagttcgcgt ccgacggcag
     3241 acaaagcgcc gccatagtcc atggtggcgc tagcataact tcgtataaag tatcctatac
     3301 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     3361 aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc
     3421 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3481 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3541 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3601 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3661 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3721 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3781 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3841 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3901 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3961 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     4021 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     4081 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     4141 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     4201 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     4261 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     4321 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     4381 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     4441 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     4501 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4561 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4621 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4681 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4741 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4801 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4861 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4921 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4981 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5041 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5101 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5161 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5221 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5281 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5341 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5401 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5461 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5521 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5581 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5641 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5701 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5761 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5821 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5881 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5941 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6001 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6061 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6121 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6181 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6241 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6301 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6361 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6421 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6481 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6541 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6601 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6661 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6721 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6781 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6841 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6901 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6961 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7021 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7081 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7141 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7201 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacg
//
