LOCUS       sequence-247071-        7241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of Ef1a-driven, cre-dependent, hChR2 variant for 
            ultrafast optogenetic control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  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 7241)
  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)-mCherry
FEATURES             Location/Qualifiers
     source          1..7241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   193..322
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        423..1601
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          654..1592
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1549..1569
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1632..1665)
                     /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(1716..1749)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1761..2468)
                     /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(2039..2058)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2308..2326
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2481..3407)
                     /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      3404..3413
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3418..3451
                     /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    3502..3535
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3560..4148
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3613..3633)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4031..4042)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4180..4656
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4379..4398)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4696..4836
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4696..4825
                     /label=AAV2 ITR
     rep_origin      4911..5366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4998..5017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5208..5229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5383..5402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5502..5524
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5562..5580)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5648..5752
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5753..5821,5822..6613)
                     /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(5971..5990)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6784..7241,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
      241 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      301 aactccatca ctaggggttc ctgcggccgc acgcgtaagc tttgcaaaga tggataaagt
      361 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa
      421 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      481 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      541 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      601 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
      661 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
      721 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt
      781 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct
      841 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg
      901 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct
      961 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg
     1021 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc
     1081 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc
     1141 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac
     1201 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga
     1261 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc
     1321 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg
     1381 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga
     1441 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt
     1501 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga
     1561 cagtggttca aagttttttt cttccatttc aggtgtcgtg aggtaccgga tcctctagag
     1621 tcgactccgg aataacttcg tataggatac tttatacgaa gttatgcaga atggtagctg
     1681 gattgtagct gctattagca atatgaaacc tcttaataac ttcgtatagc atacattata
     1741 cgaagttatg gcgcgcccta ttacttgtac agctcgtcca tgccgccggt ggagtggcgg
     1801 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
     1861 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
     1921 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
     1981 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
     2041 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
     2101 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     2161 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
     2221 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
     2281 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     2341 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     2401 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
     2461 ttgctcacgg cggccgctgg cacggctccg gcctcggctt cgtcttcgac gagagtctcg
     2521 acctcgatct ccgttccgcc gatgttcagt ttggtggttt tgcggatatc tccgtgaatc
     2581 aatatgtgct cgtggatcag gacgcgcagg tagtgtccca acaaccccca acaattttta
     2641 ctcatcagat caataatcgt gtgacctacg gtggagccat agacgctcag gacgccaaaa
     2701 ccttcgggcc ccaaaatgaa gagaattggg aacatacccc agctcacgaa aaacagccat
     2761 gccatgccgg tcacgacctg gcggcaccga ccctttggca cagtatgata accctcgata
     2821 tatgctttgg cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag acaaaagaag
     2881 atgactttaa catagccggt tgccatggcg ctggtagccc cccacacgat acacccgatg
     2941 tctgagacaa ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc ggtgaggttg
     3001 ctcaggtgga taaggatgac aggacaagtg agcagccagg ttgcatagcg cagccactgc
     3061 acccggtgtc ctgtggcaag gtagagcata gagggattct taaactcaaa aaagaactcg
     3121 agaatcacct taaccatttc aatggcgcac acatagatct cctcccagcc gcatgtagat
     3181 ttccaggttt ggtaggcata gaacatcagc agcaaaatgc tgaatcctgc tgcaagccac
     3241 tgcaggacat ttgacgcggt ctgagcgccg ttcgtgccgc gagattcaat ccatccggca
     3301 cagtaacatt gatcctcagg gaccaggacg gacccgttca ccaccacagg attagtaacg
     3361 aacaaaagtt cgcgtccgac ggcagacaaa gcgccgccat agtccatggt ggctagcata
     3421 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta
     3481 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt
     3541 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3601 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3661 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3721 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3781 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3841 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3901 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3961 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     4021 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4081 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4141 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     4201 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4261 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4321 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4381 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4441 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4501 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4561 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4621 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4681 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4741 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4801 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4861 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4921 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4981 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5041 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5101 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     5161 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5221 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5281 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5341 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5401 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5461 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5521 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5581 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5641 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5701 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5761 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5821 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5881 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5941 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6001 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6061 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6121 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6181 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6241 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6301 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6361 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6421 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6481 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6541 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6601 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7201 agggagaaag gcggacaggt atccggtaag cggcagggtc g
//
