LOCUS       sequence-362034-        7307 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 7307)
  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 7307)
  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-hsChRmine-oScarlet-WPRE
FEATURES             Location/Qualifiers
     source          1..7307
                     /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(1440..1473)
                     /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(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1593..2285)
                     /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(1868..1887)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2137..2155
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3278..3287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3292..3325
                     /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    3376..3409
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3434..4022
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3487..3507)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3905..3916)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4054..4530
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4253..4272)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4570..4710
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4570..4699
                     /label=AAV2 ITR
     rep_origin      4785..5240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4872..4891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5082..5103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5257..5276)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5376..5398
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5436..5454)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5522..5626
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5627..5695,5696..6487)
                     /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(5845..5864)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6658..7246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7147..7166
                     /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 gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgcctcatt acacctcgtt ctcgtagcag aacttgtaca gctcgtccat gccgccggtg
     1621 gagtggcggc cctcggagcg ttcgtactgt tccaccacgg tgtagtcctc gttgtgggag
     1681 gtgatgtcca acttgcggtc cacgttgtag gcgccgggca tctgcacggg cttcttggcc
     1741 ttgtaggtgg tcttgaagtc cgccaggtag cgcccgccgt ccttcaggcg cagggccatc
     1801 ttaatgtcgc ccttcagcac gccgtcctcg gggtacaacc gctcggtgga cgcttcccag
     1861 cccattgtct tcttctgcat tacggggccg tcaggaggga agttggtgcc gcggagcttc
     1921 accttgtaga tcagggtgcc gtcctccagg gaggtgtcct gggtcacggt cacggcgccg
     1981 ccgtcctcga agttcatcac gcggtcccac ttgaagccct cggggaagga ctgcttatag
     2041 tagtcgggga tgtcggcggg gtgcttggtg aaggccctgg agccgtacat gaactgaggg
     2101 gacaggatgt cccaggagaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc
     2161 tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc
     2221 atggagccct ccatgtgcac cttgaaccgc atgaactcct tgatcactgc ctcgcccttg
     2281 ctcaccacgt tgatgtcgat ctggtccagg gggatgtact cgccctcgct ggtgatcctg
     2341 ctcttggcgg ccgcagacag tctccgcagg cgctcgccat tgctgctcag cctcatatcc
     2401 agctccagct tctggaactc atcctcgtcg tgtcccagct gctccagcag ccgaaacagc
     2461 tcctcatcat aggtctttct gaaccgggcc agggcaaagc cgtaacagga cttggccacg
     2521 atgtcgcaca cgcagtgggc cacctctgtc cagttatcgt cgatccagcc gaagcctcta
     2581 gggcagatgg cccacaccag gggaaacaga atccacagca tggagaaggt aaacacggcg
     2641 aacttcagca cgtgcaggct gtgctcggcg cctgtatcct gggccagctg ggccagccgg
     2701 ctatactgct tggccacgat gctcatcacg atagagtagg cgaagataaa ccagaaacag
     2761 ccaaagccat accaggcgta ggcgccattc ctcagcctag ggtcgccctc tgcgtagaac
     2821 tcggccagca cgccagacat caggatggca aagatgatgg cgctacaaga cacgcgatag
     2881 ggggccctca gctgatacag cagatcgtac accagcattg ggcaggtcag catgtagtcg
     2941 gcatacctgt aattgccgaa gccgatgatg aaggggccca catcgccgaa gttgtggccc
     3001 agtgtgtcgg ccacgatctt gccgaagtag ctcagcatgg caaagaaatt cacaaacagg
     3061 gccaggttct gctcctcctg atagccggcg taaaaggagg ttctcttgct agggccccga
     3121 gatgtccagg actcatagcc caggtagaat gtggtgattg tcagcacgat aaagcaagac
     3181 cacctggcgc cgatggccac ggagttcagc ttggtatcca gataccagcc gtccattgtg
     3241 cccacgtaga acatctggtc ggtgcctggt gcgtgtgcca tggtggctag cataacttcg
     3301 tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt
     3361 agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg aattcgatat
     3421 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3481 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3541 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3601 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3661 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3721 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3781 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3841 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3901 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     3961 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4021 gcatcgatac cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca
     4081 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     4141 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     4201 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     4261 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     4321 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     4381 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     4441 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     4501 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
     4561 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4621 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4681 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4741 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4801 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4861 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4921 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4981 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5041 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5101 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5161 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5221 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5281 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5341 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5401 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5461 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5521 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5581 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5641 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5701 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5761 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5821 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5881 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5941 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6001 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6061 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6121 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6181 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6241 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6301 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6361 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6421 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6481 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6541 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6601 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6661 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6721 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6781 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6841 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6901 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6961 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7021 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7081 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7141 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7201 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7261 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
