LOCUS       sequence-362061-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  AUTHORS   Kishi KE, Kim YS, Fukuda M, Inoue M, Kusakizako T, Wang PY, 
            Ramakrishnan C, Byrne EFX, Thadhani E, Paggi JM, Matsui TE, 
            Yamashita K, Nagata T, Konno M, Quirin S, Lo M, Benster T, Uemura T,
            Liu K, Shibata M, Nomura N, Iwata S, Nureki O, Dror RO, Inoue K, 
            Deisseroth K, Kato HE
  TITLE     Structural basis for channel conduction in the pump-like 
            channelrhodopsin ChRmine.
  JOURNAL   Cell. 2022 Feb 17;185(4):672-689.e23. doi: 
            10.1016/j.cell.2022.01.007. Epub 2022 Feb 2.
  PUBMED    35114111
REFERENCE   2  (bases 1 to 7283)
  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: 
            "10.1016/j.cell.2022.01.007"; journalName: "Cell"; date: 
            "2022-02-17- 17"; volume: "185"; issue: "4"; pages: "672-689.e23"
COMMENT     Sequence Label: pAAV-Ef1a DIO-ChRmine-oScarlet-WPRE
FEATURES             Location/Qualifiers
     source          1..7283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1416..1449)
                     /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(1500..1533)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1569..2261)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1844..1863)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2113..2131
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3254..3263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3268..3301
                     /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    3352..3385
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3410..3998
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3463..3483)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3881..3892)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4030..4506
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4229..4248)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4546..4686
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4546..4675
                     /label=AAV2 ITR
     rep_origin      4761..5216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4848..4867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5058..5079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5233..5252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5352..5374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5412..5430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5498..5602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5603..5671,5672..6463)
                     /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(5821..5840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6634..7222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7123..7142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gatccataac ttcgtatagg atactttata
     1441 cgaagttatg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa
     1501 taacttcgta tagcatacat tatacgaagt tatggcgcgc cctattacac ctcgttctcg
     1561 tagcagaact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggagcgttcg
     1621 tactgttcca ccacggtgta gtcctcgttg tgggaggtga tgtccaactt gcggtccacg
     1681 ttgtaggcgc cgggcatctg cacgggcttc ttggccttgt aggtggtctt gaagtccgcc
     1741 aggtagcgcc cgccgtcctt caggcgcagg gccatcttaa tgtcgccctt cagcacgccg
     1801 tcctcggggt acaaccgctc ggtggacgct tcccagccca ttgtcttctt ctgcattacg
     1861 gggccgtcag gagggaagtt ggtgccgcgg agcttcacct tgtagatcag ggtgccgtcc
     1921 tccagggagg tgtcctgggt cacggtcacg gcgccgccgt cctcgaagtt catcacgcgg
     1981 tcccacttga agccctcggg gaaggactgc ttatagtagt cggggatgtc ggcggggtgc
     2041 ttggtgaagg ccctggagcc gtacatgaac tgaggggaca ggatgtccca ggagaagggc
     2101 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     2161 tcgccctcgc cctcgatctc gaactcgtgg ccgttcatgg agccctccat gtgcaccttg
     2221 aaccgcatga actccttgat cactgcctcg cccttgctca ccacgttgat gtcgatctgg
     2281 tccaggggga tgtactcgcc ctcgctggtg atcctgctct tggcggccgc agacagtctc
     2341 cgcaggcgct cgccattgct gctcagcctc atatccagct ccagcttctg gaactcatcc
     2401 tcgtcgtgtc ccagctgctc cagcagccga aacagctcct catcataggt ctttctgaac
     2461 cgggccaggg caaagccgta acaggacttg gccacgatgt cgcacacgca gtgggccacc
     2521 tctgtccagt tatcgtcgat ccagccgaag cctctagggc agatggccca caccagggga
     2581 aacagaatcc acagcatgga gaaggtaaac acggcgaact tcagcacgtg caggctgtgc
     2641 tcggcgcctg tatcctgggc cagctgggcc agccggctat actgcttggc cacgatgctc
     2701 atcacgatag agtaggcgaa gataaaccag aaacagccaa agccatacca ggcgtaggcg
     2761 ccattcctca gcctagggtc gccctctgcg tagaactcgg ccagcacgcc agacatcagg
     2821 atggcaaaga tgatggcgct acaagacacg cgataggggg ccctcagctg atacagcaga
     2881 tcgtacacca gcattgggca ggtcagcatg tagtcggcat acctgtaatt gccgaagccg
     2941 atgatgaagg ggcccacatc gccgaagttg tggcccagtg tgtcggccac gatcttgccg
     3001 aagtagctca gcatggcaaa gaaattcaca aacagggcca ggttctgctc ctcctgatag
     3061 ccggcgtaaa aggaggttct cttgctaggg ccccgagatg tccaggactc atagcccagg
     3121 tagaatgtgg tgattgtcag cacgataaag caagaccagt gggcgccgat ggccacggag
     3181 ttcagcttgg tatccagata ccagccgtcc attgtgccca cgtagaacat ctggtcggtg
     3241 cctggtgcgt gtgccatggt ggctagcata acttcgtata aagtatccta tacgaagtta
     3301 tttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg
     3361 tataatgtat gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct
     3421 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3481 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3541 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3601 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3661 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3721 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3781 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     3841 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3901 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3961 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     4021 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     4081 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     4141 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     4201 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     4261 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     4321 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     4381 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     4441 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     4501 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     4561 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4621 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     4681 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4741 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4801 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     4861 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4921 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     4981 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5041 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     5101 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     5161 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     5221 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5401 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5461 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5521 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5581 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5641 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5701 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5761 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5821 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5881 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5941 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6001 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6061 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6121 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6181 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6241 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6301 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6361 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6421 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6481 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6541 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6601 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6661 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6721 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6781 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6841 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6901 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6961 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7021 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7081 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7141 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7201 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7261 ttgctggcct tttgctcaca tgt
//
