LOCUS       sequence-196786-        7243 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 7243)
  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 7243)
  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)-mCherry
FEATURES             Location/Qualifiers
     source          1..7243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   61..190
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        291..1469
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          522..1460
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1417..1437
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1500..1533)
                     /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(1584..1617)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1629..2336)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1907..1926)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2176..2194
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2349..3275)
                     /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      3272..3281
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3288..3321
                     /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    3372..3405
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3430..4018
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3483..3503)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3901..3912)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4050..4526
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4249..4268)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4566..4706
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4566..4695
                     /label=AAV2 ITR
     rep_origin      4781..5236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4868..4887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5078..5099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5253..5272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5372..5394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5432..5450)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5518..5622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5623..5691,5692..6483)
                     /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(5841..5860)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6654..7242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7143..7162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
       61 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
      121 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      181 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      241 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      301 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      361 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      421 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      481 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      541 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      601 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      661 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      721 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      781 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      841 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      901 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      961 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     1021 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1081 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1141 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1201 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1261 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1321 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1381 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1441 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1501 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1561 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1621 gcgccctatt acttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt
     1681 tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg
     1741 acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca
     1801 gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg
     1861 ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt
     1921 acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg
     1981 tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg
     2041 cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg
     2101 tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag
     2161 ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg
     2221 ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc
     2281 ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt gctcacggcg
     2341 gccgctggca cggctccggc ctcggcttcg tcttcgacga gagtctcgac ctcgatctcc
     2401 gttccgccga tgttcagttt ggtggttttg cggatatctc cgtgaatcaa tatgtgctcg
     2461 tggatcagga cgcgcaggta gtgtcccaac aacccccaac aatttttact catcagatca
     2521 ataatcgtgt gacctacggt ggagccatag acgctcagga cgccaaaacc ttcgggcccc
     2581 aaaatgaaga gaattgggaa cataccccag ctcacgaaaa acagccatgc catgccggtc
     2641 acgacctggc ggcaccgacc ctttggcaca gtatgataac cctcgatata tgctttggcg
     2701 gcgtgaaaaa atgtgttcgc gccatagcac aatccaagac aaaagaagat gactttaaca
     2761 tagccggttg ccatggcgct ggtagccccc cacacgatag tcccgatggc tgagacaagg
     2821 agtcccatgg ttctcctgct gtagtcgttg ctcaggccgg tgaggttgct caggtggata
     2881 aggatgacag gagaagtgag cagccactct gcatagcgca gccactgcac ccggtgtcct
     2941 gtggcaaggt agagcataga gggattctta aactcaaaaa agaactcgag aatcacctta
     3001 accatttcaa tggcgcacac atagatctcc tcccagccgc atgtagattt ccaggtttgg
     3061 taggcataga acatcagcag caaaatgctg aatcctgctg caagccactg caggacattt
     3121 gacgcggtct gagcgccgtt cgtgccgcga gattcaatcc atccggcaca gtaacattga
     3181 tcctcaggga ccaggacgga cccgttcacc accacaggat tagtaacgaa caaaagttcg
     3241 cgtccgacgg cagacaaagc gccgccatag tccatggtgg cgctagcata acttcgtata
     3301 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
     3361 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
     3421 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     3481 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     3541 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     3601 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     3661 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     3721 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3781 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     3841 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3901 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     3961 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     4021 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     4081 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     4141 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     4201 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     4261 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     4321 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     4381 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     4441 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     4501 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     4561 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4621 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4681 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4741 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4801 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4861 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4921 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4981 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5041 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5101 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5161 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5221 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5281 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5341 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5401 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5461 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5521 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5581 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5641 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5701 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5761 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5821 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5881 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5941 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6001 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6061 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6121 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6181 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6241 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6301 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6361 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6421 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6481 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6541 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6781 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaa
//
