LOCUS       sequence-196788-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of EF1a-driven, cre-dependent, hChR2 variant CheTA 
            2.0 for ultrafast optogenetic control..
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(E123A)-EYFP
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   47..176
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        277..1455
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          508..1446
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1403..1423
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1486..1519)
                     /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(1570..1603)
                     /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(1615..2328,2329..2331,2332..2334))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1660..1681)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2009..2028
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2268..2289
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2331..2340
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2350..3276)
                     /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
                     PSMLYLATGHRVQWLRYAAWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3273..3282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3287..3320
                     /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    3371..3404
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3429..4017
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3482..3502)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3900..3911)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4049..4525
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4248..4267)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4565..4705
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4565..4694
                     /label=AAV2 ITR
     rep_origin      4780..5235
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4867..4886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5077..5098
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5252..5271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5371..5393
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5431..5449)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5517..5621
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5622..5690,5691..6482)
                     /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(5840..5859)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6653..7241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7142..7161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg
       61 cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc
      121 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg
      181 ccgcacgcgt aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc
      241 taatggacct tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca
      301 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
      361 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
      421 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
      481 cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc
      541 ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg
      601 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
      661 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
      721 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
      781 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
      841 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
      901 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
      961 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     1021 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     1081 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     1141 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     1201 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     1261 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     1321 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     1381 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     1441 tttcaggtgt cgtgaggtac cggatcctct agagtcgact ccggaataac ttcgtatagg
     1501 atactttata cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga
     1561 aacctcttaa taacttcgta tagcatacat tatacgaagt tatggcgcgc cctattactt
     1621 gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca gcaggaccat
     1681 gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tggtagctca ggtagtggtt
     1741 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
     1801 ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga tgccgttctt
     1861 ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca gcttgtgccc
     1921 caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt
     1981 gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat
     2041 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
     2101 gtggtcgggg tagcgggcga agcactgcag gccgtagccg aaggtggtca cgagggtggg
     2161 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt
     2221 ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag
     2281 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc
     2341 ggccgctggc acggctccgg cctcggcttc gtcttcgacg agagtctcga cctcgatctc
     2401 cgttccgccg atgttcagtt tggtggtttt gcggatatct ccgtgaatca atatgtgctc
     2461 gtggatcagg acgcgcaggt agtgtcccaa caacccccaa caatttttac tcatcagatc
     2521 aataatcgtg tgacctacgg tggagccata gacgctcagg acgccaaaac cttcgggccc
     2581 caaaatgaag agaattggga acatacccca gctcacgaaa aacagccatg ccatgccggt
     2641 cacgacctgg cggcaccgac cctttggcac agtatgataa ccctcgatat atgctttggc
     2701 ggcgtgaaaa aatgtgttcg cgccatagca caatccaaga caaaagaaga tgactttaac
     2761 atagccggtt gccatggcgc tggtagcccc ccacacgata gtcccgatgt ctgagacaag
     2821 gagtcccatg gttctcctgc tgtagtcgtt gctcaggccg gtgaggttgc tcaggtggat
     2881 aaggatgaca ggacaagtga gcagccaggc tgcatagcgc agccactgca cccggtgtcc
     2941 tgtggcaagg tagagcatag agggattctt aaactcaaaa aagaactcga gaatcacctt
     3001 aaccatttca atggcgcaca catagatctc ctcccagccg catgtagatt tccaggtttg
     3061 gtaggcatag aacatcagca gcaaaatgct gaatcctgct gcaagccact gcaggacatt
     3121 tgacgcggtc tgagcgccgt tcgtgccgcg agattcaatc catccggcac agtaacattg
     3181 atcctcaggg accaggacgg acccgttcac caccacagga ttagtaacga acaaaagttc
     3241 gcgtccgacg gcagacaaag cgccgccata gtccatggtg gctagcataa cttcgtataa
     3301 agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat
     3361 ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatgaattc gatatcaagc
     3421 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3481 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3541 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3601 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3661 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3721 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3781 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3841 ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3901 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3961 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     4021 gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc
     4081 tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag
     4141 ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt
     4201 atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc
     4261 aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc
     4321 gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag
     4381 ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa
     4441 ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg
     4501 aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg
     4561 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4621 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4681 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4741 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4801 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4861 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4921 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4981 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5041 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5101 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5161 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5221 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5281 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5341 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5401 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5461 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5521 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5581 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5641 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5701 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5761 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5821 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5881 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5941 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6001 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6061 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6121 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6181 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6241 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6301 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6361 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6421 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6481 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6541 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6601 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6661 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6721 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6781 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6841 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6901 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6961 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7021 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7081 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7141 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7201 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcg
//
