LOCUS       sequence-257680-        6941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV-mediated expression of ChrimsonR-GFP under the EF1-alpha-1.1 
            promoter in frt/reversed (Flp-dependent) manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6941)
  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 6941)
  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-EF1-alpha-1.1-FLPX-rc [ChrimsonR-GFP]
FEATURES             Location/Qualifiers
     source          1..6941
                     /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        145..1323
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          376..1314
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1271..1291
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1330..1363
                     /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(1457..2170,2171..2173))
                     /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(1460..2170,2171..2173))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1502..1523)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1851..1870
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2110..2131
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2192..3241)
                     /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      3238..3247
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3265..3298)
                     /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    3388..3976
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3441..3461)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3859..3870)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   4204..4344
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4204..4333
                     /label=AAV2 ITR
     rep_origin      4419..4874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4506..4525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4716..4737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4891..4910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5010..5032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5070..5088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5156..5260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5261..5329,5330..6121)
                     /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(5479..5498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6292..6880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6781..6800
                     /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 gtcgacttaa ttaaggctcc ggtgcccgtc agtgggcaga gcgcacatcg
      181 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
      241 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
      301 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
      361 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
      421 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
      481 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
      541 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
      601 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
      661 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
      721 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
      781 tgttcgacga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
      841 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
      901 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
      961 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     1021 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     1081 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     1141 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     1201 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     1261 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     1321 tgagctagcg aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1381 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1441 gaacttcgaa ttctcattac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1501 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
     1561 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     1621 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
     1681 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
     1741 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     1801 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     1861 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     1921 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     1981 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg caaatgaact
     2041 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     2101 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     2161 cgcccttgct cactgctact accggtgccg ccactgtgtc ctcgtcctcc tcctccacga
     2221 actcttccac ttccacctcc tcgcctccga tctccatctt ggtggtcttc cggatgtcgc
     2281 cgtggatcag gatgtgctcg tggatcttga tcctcaggtg gtgggccagg aaggtccaaa
     2341 actccttggc gatgatgtcg cagatgctgt ggccgatgct gttggcgtaa gggctcagct
     2401 tcagcagtcc ttctggtccc actgcccaga ggattgggta gctgccccaa gaggcgaagt
     2461 aagcgtaggc catcagcttc acgaccatgc ggcaatggcc tttaggcacg ctgtggttgg
     2521 cttccacgta gcacttggcg gcctggaagt acatgtagcc gccgtagatg caagacacga
     2581 tatacagcag ccacttgagc caatcggtag ccagtcctgc ggccatgccg aacacgatca
     2641 ttcccacgca agacacgatc aggcccatgg tccgcttgct gtagtcgttc ttcaggccgc
     2701 tcaggttgct cagtctgatc aggatcacgg ggcaagacag cagccactcg aagtagcgca
     2761 ggcaataggc gtggttgccg gtagacaggt acactgtggc ggggctgctg aactccttga
     2821 agatctccag ggtcacgaac agcacctcga cgcaacagac gtagacctcc tcccaaccgc
     2881 aagtggcctt ccaggcgctg aagccgtaga atgtcagcag ggcgatggcg atgctgaaag
     2941 caatccactg gcagacctgg gcgccgatct tttctcctgg tgtgcctctg ctgtgcagcc
     3001 aagcctgctt cttctccact cccacgacca ggtagccgcc tgtggcctcc acgaagcaat
     3061 atccgtatcc ggcgtcgctc agtccgtaag aagggtcaca ccaccactcg gtgctgaagc
     3121 actcgcctgt aggtgtcatt cctccacagc ccatgtcctc gtggtggtag gggttaggcc
     3181 aagggttgat gcctccggcg gcaaacagag atctggtggc gctgctgatc agctcagcca
     3241 tggtggcgga tccggcggcg cgccgaagtt cctatacttt ctagagaata ggaacttctt
     3301 gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaga agttcctata
     3361 ccttttgaag aataggaact tctccggaat caacctctgg attacaaaat ttgtgaaaga
     3421 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     3481 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     3541 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     3601 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     3661 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     3721 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     3781 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     3841 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3901 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3961 tgggccgcct ccccgccttg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4021 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4081 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4141 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4201 ggcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4261 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4321 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4381 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4621 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4741 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4861 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4921 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4981 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5041 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5101 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5161 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5221 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5281 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5341 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5401 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5461 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5521 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5581 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5641 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5701 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5761 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5821 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5881 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5941 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6001 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6061 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6121 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6421 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6901 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
