LOCUS       sequence-397078-        7066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses bicistronically soma-targeted ChrimsonR in frame with EGFP
            and the optimized PdCO under control of human synapsin1 promotor 
            after Cre-dependent recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7066)
  AUTHORS   Wietek J, Nozownik A, Pulin M, Saraf-Sinik I, Matosevich N, 
            Gowrishankar R, Gat A, Malan D, Brown BJ, Dine J, Imambocus BN, Levy
            R, Sauter K, Litvin A, Regev N, Subramaniam S, Abrera K, Summarli D,
            Goren EM, Mizrachi G, Bitton E, Benjamin A, Copits BA, Sasse P, Rost
            BR, Schmitz D, Bruchas MR, Soba P, Oren-Suissa M, Nir Y, Wiegert JS,
            Yizhar O
  TITLE     A bistable inhibitory optoGPCR for multiplexed optogenetic control 
            of neural circuits.
  JOURNAL   Nat Methods. 2024 May 29. doi: 10.1038/s41592-024-02285-8.
  PUBMED    38811857
REFERENCE   2  (bases 1 to 7066)
  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"; journalName: "Nat 
            Methods. 2024 May 29. doi: 10.1038/s41592-024-02285-8."
COMMENT     Sequence Label: pAAV_hSyn-SIO-stChrimsonR-EGFP-P2A-PdCO-miniWPRE
FEATURES             Location/Qualifiers
     source          1..7066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(506..1555)
                     /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"
     protein_bind    1562..1595
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1658..1678)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   2053..2193
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2053..2182
                     /label=AAV2 ITR
     rep_origin      2268..2723
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2355..2374)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2565..2586
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2740..2759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2859..2881
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2919..2937)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3005..3109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3110..3178,3179..3970)
                     /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(3328..3347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4141..4729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4630..4649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4791..4920
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        4965..5412
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(5426..5459)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(6558..6614)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(6843..7066,1..484,485..487))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             complement(join(6843..7066,1..484,485..487))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(6882..6903)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcaaatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcactgc tactaccggt gccgccactg tgtcctcgtc ctcctcctcc acgaactctt
      541 ccacttccac ctcctcgcct ccgatctcca tcttggtggt cttccggatg tcgccgtgga
      601 tcaggatgtg ctcgtggatc ttgatcctca ggtggtgggc caggaaggtc caaaactcct
      661 tggcgatgat gtcgcagatg ctgtggccga tgctgttggc gtaagggctc agcttcagca
      721 gtccttctgg tcccactgcc cagaggattg ggtagctgcc ccaagaggcg aagtaagcgt
      781 aggccatcag cttcacgacc atgcggcaat ggcctttagg cacgctgtgg ttggcttcca
      841 cgtagcactt ggcggcctgg aagtacatgt agccgccgta gatgcaagac acgatataca
      901 gcagccactt gagccaatcg gtagccagtc ctgcggccat gccgaacacg atcattccca
      961 cgcaagacac gatcaggccc atggtccgct tgctgtagtc gttcttcagg ccgctcaggt
     1021 tgctcagtct gatcaggatc acggggcaag acagcagcca ctcgaagtag cgcaggcaat
     1081 aggcgtggtt gccggtagac aggtacactg tggcggggct gctgaactcc ttgaagatct
     1141 ccagggtcac gaacagcacc tcgacgcaac agacgtagac ctcctcccaa ccgcaagtgg
     1201 ccttccaggc gctgaagccg tagaatgtca gcagggcgat ggcgatgctg aaagcaatcc
     1261 actggcagac ctgggcgccg atcttttctc ctggtgtgcc tctgctgtgc agccaagcct
     1321 gcttcttctc cactcccacg accaggtagc cgcctgtggc ctccacgaag caatatccgt
     1381 atccggcgtc gctcagtccg taagaagggt cacaccacca ctcggtgctg aagcactcgc
     1441 ctgtaggtgt cattcctcca cagcccatgt cctcgtggtg gtaggggtta ggccaagggt
     1501 tgatgcctcc ggcggcaaac agagatctgg tggcgctgct gatcagctca gccatggatc
     1561 ctaccgttcg tataatgtat gctatacgaa gttataagct tgataatcaa cctctggatt
     1621 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     1681 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     1741 cctccttgta taaatcctgg ttagttcttg ccacggcgga actcatcgcc gcctgccttg
     1801 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg tttatttgtg
     1861 aaatttgtga tgctattgct ttatttgtaa ccatctagct ttatttgtga aatttgtgat
     1921 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1981 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag cacgtgcgga
     2041 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     2101 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     2161 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     2221 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     2281 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2341 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2401 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2461 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     2521 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2581 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     2641 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2701 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     2761 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2821 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2881 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     2941 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     3001 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3061 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3121 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3181 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     3241 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     3301 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     3361 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     3421 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     3481 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     3541 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     3601 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     3661 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     3721 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     3781 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     3841 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     3901 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     3961 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4021 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4081 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4141 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4201 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4261 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4321 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4381 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4441 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4501 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4561 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4621 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4681 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4741 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
     4801 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
     4861 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     4921 gcggccgcac gcgtgtgtct agactgcaga gggccctgcg tatgagtgca agtgggtttt
     4981 aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga cccactggac
     5041 aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg gaggggaaac
     5101 aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt gccttcgccc
     5161 ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc
     5221 ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc cgccggccca
     5281 gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc
     5341 ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc
     5401 ctgagagcgc agtcgagaag gtacctaccg ttcgtatagc atacattata cgaagttatg
     5461 aattcttaag caggagcaac ctgggaagtc tcggtcgtcg cggcgacttg attcgtggct
     5521 gtaccaccag ttgctctctc actctcggag ttcaggtcct ctactcccat tccgagaaga
     5581 actttaaggc tccgcctgaa ttgtttgttc atgaacacat agataagagg gttatacatc
     5641 gtagaagact ttgcaaacat ggcaggaatc actgtggcca ctggatggag caggtcttta
     5701 gggccgaagc tagcgagcat gcagataaca gcgtagggac tccaggcaag catgaacagg
     5761 ctaaccatca gcagcgtcat gcgcagtact tcgcgttctg ctttggcaac agaaccgcct
     5821 gttttcgcta ccttccgaac cttatcgtag atcagcccgt atgacacgac gataatgaga
     5881 acagggacaa gaaagcagaa gatgaaaata gcaaacacgt aggacttatc ggatgtttca
     5941 tcggaaagcc agtcgatact acaccaagtg gccaaacctt ccggtatgta gtgattccac
     6001 ccaaggagag gcgcgagtgt gaatgccagg gagtagatcc aaatgaaagc catcgccgcc
     6061 acgcccatcc gcgttgtcaa acctgtgaag gagccgagac gcagcacggc cagacaacga
     6121 acaaaagcaa taacgctcag ggtcatcagt gccgcgagcc cgccgaggta tgttatgaag
     6181 ccgtaccatt tgcagaaacc gtcttcttct cgcaacaggg ttttgcgagc tgtaataatg
     6241 aacttaaatg cggtgccgaa aactgcaacc aacagatccc cgagagcgag acttacatac
     6301 aggaagttat tggggctcct caactttctg tctttgataa agaggaccaa caccaaaacg
     6361 ttgagactga ctcccagaca agcaatgaag aaaaggtaag ctgcacaaat gttgtaatcg
     6421 gtggctgtga aggcattgta gatgtaatga tttccgccgt ccgtttccgc attaaggttt
     6481 tggaaatacg tgctgttgat tggggtatcc atcaactcgg tgacaggatt tggaatcgtc
     6541 aagttctccc cgtccatagg tccagggttc tcctccacgt ctccagcctg cttcagcagg
     6601 ctgaagttag tagctccgct accgtacgaa aaccgcgtag cctctggaaa atcggtggcg
     6661 catgagataa aagagtcaat agaactcatg gacctcatgt cgatcacgcc ctctgttctt
     6721 gcaggaagag gtgccacagg ggagggcatt gaggacattt ccagctcctc agggggttta
     6781 ctctgggggg ccatctcttt ggtgttcaga ataggctggg actgcccgta cgcgccggcg
     6841 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     6901 atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
     6961 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
     7021 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttca
//
