LOCUS       sequence-362033-        7478 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 7478)
  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 7478)
  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-Kv2.1-WPRE
FEATURES             Location/Qualifiers
     source          1..7478
                     /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(1764..2456)
                     /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(2039..2058)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2308..2326
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3449..3458
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3463..3496
                     /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    3547..3580
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3605..4193
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3658..3678)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4076..4087)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4225..4701
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4424..4443)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4741..4881
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4741..4870
                     /label=AAV2 ITR
     rep_origin      4956..5411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5043..5062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5253..5274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5428..5447)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5547..5569
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5607..5625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5693..5797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5798..5866,5867..6658)
                     /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(6016..6035)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6829..7417
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7318..7337
                     /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 tagcagaaga atctggtggc ttcagggaag tccgtggcac agctgatgaa gctgtcaatg
     1621 ctggacatgc tccgcatgtc gatgacgccc tccgtgcgtg cgggcagagg ggccacgggg
     1681 ctgggcatgc tgctcatctc cagctcctct ggaggcttgc tctgcggggc catctccttg
     1741 gtgttgagga tgggctggga ctgcttgtac agctcgtcca tgccgccggt ggagtggcgg
     1801 ccctcggagc gttcgtactg ttccaccacg gtgtagtcct cgttgtggga ggtgatgtcc
     1861 aacttgcggt ccacgttgta ggcgccgggc atctgcacgg gcttcttggc cttgtaggtg
     1921 gtcttgaagt ccgccaggta gcgcccgccg tccttcaggc gcagggccat cttaatgtcg
     1981 cccttcagca cgccgtcctc ggggtacaac cgctcggtgg acgcttccca gcccattgtc
     2041 ttcttctgca ttacggggcc gtcaggaggg aagttggtgc cgcggagctt caccttgtag
     2101 atcagggtgc cgtcctccag ggaggtgtcc tgggtcacgg tcacggcgcc gccgtcctcg
     2161 aagttcatca cgcggtccca cttgaagccc tcggggaagg actgcttata gtagtcgggg
     2221 atgtcggcgg ggtgcttggt gaaggccctg gagccgtaca tgaactgagg ggacaggatg
     2281 tcccaggaga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     2341 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt catggagccc
     2401 tccatgtgca ccttgaaccg catgaactcc ttgatcactg cctcgccctt gctcaccacg
     2461 ttgatgtcga tctggtccag ggggatgtac tcgccctcgc tggtgatcct gctcttggcg
     2521 gccgcagaca gtctccgcag gcgctcgcca ttgctgctca gcctcatatc cagctccagc
     2581 ttctggaact catcctcgtc gtgtcccagc tgctccagca gccgaaacag ctcctcatca
     2641 taggtctttc tgaaccgggc cagggcaaag ccgtaacagg acttggccac gatgtcgcac
     2701 acgcagtggg ccacctctgt ccagttatcg tcgatccagc cgaagcctct agggcagatg
     2761 gcccacacca ggggaaacag aatccacagc atggagaagg taaacacggc gaacttcagc
     2821 acgtgcaggc tgtgctcggc gcctgtatcc tgggccagct gggccagccg gctatactgc
     2881 ttggccacga tgctcatcac gatagagtag gcgaagataa accagaaaca gccaaagcca
     2941 taccaggcgt aggcgccatt cctcagccta gggtcgccct ctgcgtagaa ctcggccagc
     3001 acgccagaca tcaggatggc aaagatgatg gcgctacaag acacgcgata gggggccctc
     3061 agctgataca gcagatcgta caccagcatt gggcaggtca gcatgtagtc ggcatacctg
     3121 taattgccga agccgatgat gaaggggccc acatcgccga agttgtggcc cagtgtgtcg
     3181 gccacgatct tgccgaagta gctcagcatg gcaaagaaat tcacaaacag ggccaggttc
     3241 tgctcctcct gatagccggc gtaaaaggag gttctcttgc tagggccccg agatgtccag
     3301 gactcatagc ccaggtagaa tgtggtgatt gtcagcacga taaagcaaga ccagtgggcg
     3361 ccgatggcca cggagttcag cttggtatcc agataccagc cgtccattgt gcccacgtag
     3421 aacatctggt cggtgcctgg tgcgtgtgcc atggtggcta gcataacttc gtataaagta
     3481 tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg
     3541 caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat
     3601 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3661 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3721 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3781 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3841 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     3901 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     3961 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4021 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4081 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4141 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     4201 ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     4261 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     4321 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     4381 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     4441 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     4501 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     4561 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     4621 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     4681 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     4741 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4801 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4861 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     4921 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     4981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     5041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5101 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5161 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5281 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5401 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5461 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5521 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5581 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5641 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5761 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5821 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5881 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5941 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6001 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6061 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6121 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6181 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6241 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6301 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6361 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6421 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6481 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6541 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6601 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6661 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6721 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6781 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6841 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6901 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6961 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7021 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7081 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7141 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7201 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7261 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7321 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7381 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7441 acggttcctg gccttttgct ggccttttgc tcacatgt
//
