LOCUS       sequence-257600-        6690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of ChrimsonR-GFP under the CAG promoter in 
            frt/reversed (Flp-dependent) manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6690)
  AUTHORS   Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK,
            Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, 
            Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, 
            Constantine-Paton M, Wong GK, Boyden ES
  TITLE     Independent optical excitation of distinct neural populations.
  JOURNAL   Nat Methods. 2014 Mar;11(3):338-46. doi: 10.1038/nmeth.2836. Epub 
            2014 Feb 9.
  PUBMED    24509633
REFERENCE   2  (bases 1 to 6690)
  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.2836"; journalName: "Nat Methods"; date: "2014-03"; 
            volume: "11"; issue: "3"; pages: "338-46"
COMMENT     Sequence Label: AAV-CAG-FLPX-rc [ChrimsonR-GFP]
FEATURES             Location/Qualifiers
     source          1..6690
                     /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        387..664
                     /label=chicken beta-actin promoter
     primer_bind     943..965
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1027..1046
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1079..1112
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(join(1206..1919,1920..1922))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1209..1919,1920..1922))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1251..1272)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1600..1619
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1859..1880
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1941..2990)
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     regulatory      2987..2996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3008..3041)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3131..3719
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3184..3204)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3602..3613)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3727..3951
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3953..4093
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3953..4082
                     /label=AAV2 ITR
     rep_origin      4168..4623
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4255..4274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4465..4486
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4640..4659)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4759..4781
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4819..4837)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4905..5009
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5010..5078,5079..5870)
                     /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(5228..5247)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6041..6629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6530..6549
                     /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 gtcgacccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccrggs ggggsggggs ggggsgrggg
      541 gsggggsggg gsgrggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc
      901 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
      961 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1021 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgaattcga
     1081 agttcctatt ctctagaaag tataggaact tcgcagaatg gtagctggat tgtagctgct
     1141 attagcaata tgaaacctct tagaagttcc tattcttcaa aaggtatagg aacttcgcta
     1201 gctcattact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1261 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     1321 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1381 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1441 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1501 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1561 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1621 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     1681 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     1741 acgagggtgg gccagggcac gggcagcttg ccggtggtgc aaatgaactt cagggtcagc
     1801 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     1861 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     1921 actgctacta ccggtgccgc cactgtgtcc tcgtcctcct cctccacgaa ctcttccact
     1981 tccacctcct cgcctccgat ctccatcttg gtggtcttcc ggatgtcgcc gtggatcagg
     2041 atgtgctcgt ggatcttgat cctcaggtgg tgggccagga aggtccaaaa ctccttggcg
     2101 atgatgtcgc agatgctgtg gccgatgctg ttggcgtaag ggctcagctt cagcagtcct
     2161 tctggtccca ctgcccagag gattgggtag ctgccccaag aggcgaagta agcgtaggcc
     2221 atcagcttca cgaccatgcg gcaatggcct ttaggcacgc tgtggttggc ttccacgtag
     2281 cacttggcgg cctggaagta catgtagccg ccgtagatgc aagacacgat atacagcagc
     2341 cacttgagcc aatcggtagc cagtcctgcg gccatgccga acacgatcat tcccacgcaa
     2401 gacacgatca ggcccatggt ccgcttgctg tagtcgttct tcaggccgct caggttgctc
     2461 agtctgatca ggatcacggg gcaagacagc agccactcga agtagcgcag gcaataggcg
     2521 tggttgccgg tagacaggta cactgtggcg gggctgctga actccttgaa gatctccagg
     2581 gtcacgaaca gcacctcgac gcaacagacg tagacctcct cccaaccgca agtggccttc
     2641 caggcgctga agccgtagaa tgtcagcagg gcgatggcga tgctgaaagc aatccactgg
     2701 cagacctggg cgccgatctt ttctcctggt gtgcctctgc tgtgcagcca agcctgcttc
     2761 ttctccactc ccacgaccag gtagccgcct gtggcctcca cgaagcaata tccgtatccg
     2821 gcgtcgctca gtccgtaaga agggtcacac caccactcgg tgctgaagca ctcgcctgta
     2881 ggtgtcattc ctccacagcc catgtcctcg tggtggtagg ggttaggcca agggttgatg
     2941 cctccggcgg caaacagaga tctggtggcg ctgctgatca gctcagccat ggtggcggcg
     3001 gcgcgccgaa gttcctatac tttctagaga ataggaactt cttgccttaa cccagaaatt
     3061 atcactgtta ttctttagaa tggtgcaaag agaagttcct ataccttttg aagaatagga
     3121 acttctccgg aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc
     3541 atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
     3721 tggatcctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3781 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3841 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3901 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcaggaaccc
     3961 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4021 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4081 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4141 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4201 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4261 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4321 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4381 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4441 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     4501 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     4561 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4621 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4681 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4741 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4801 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4861 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4921 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4981 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5041 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5101 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5161 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5221 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5281 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5341 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5401 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5461 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5521 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5581 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5641 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5701 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5761 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5821 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5881 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5941 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6001 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6061 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6121 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6181 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6241 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6301 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6361 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6421 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6481 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6541 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6601 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6661 tggccttttg ctggcctttt gctcacatgt
//
