LOCUS       sequence-397077-        6915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses bicistronically soma-targeted ChrimsonR in frame with EGFP
            and the optimized PdCO under control of minimal CamKIIa promotor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  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 6915)
  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-CamKIIa(0.4)-stChrimsonR-EGFP-P2A-PdCO-WPRE
FEATURES             Location/Qualifiers
     source          1..6915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(213..232)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     560..581
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             849..905
                     /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"
     misc_feature    2004..2407
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     polyA_signal    2414..2462
                     /note="synthetic polyadenylation signal"
     repeat_region   2492..2632
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2492..2621
                     /label=AAV2 ITR
     rep_origin      2707..3162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2794..2813)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3004..3025
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3179..3198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3298..3320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3358..3376)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3444..3548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3549..3617,3618..4409)
                     /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(3767..3786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4580..5168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5069..5088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5230..5359
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             5757..6806
                     /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"
     CDS             join(6825..6827,6828..6915,1..620)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(6825..6827,6828..6915,1..620)
                     /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(6867..6888)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatt tgcaccaccg
       61 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      121 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      181 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      241 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      301 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
      361 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
      421 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
      481 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
      541 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
      601 tcggcatgga cgagctgtac aacgccggcg cgtacgggca gtcccagcct attctgaaca
      661 ccaaagagat ggccccccag agtaaacccc ctgaggagct ggaaatgtcc tcaatgccct
      721 cccctgtggc acctcttcct gcaagaacag agggcgtgat cgacatgagg tccatgagtt
      781 ctattgactc ttttatctca tgcgccaccg attttccaga ggctacgcgg ttttcgtacg
      841 gtagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg
      901 gacctatgga cggggagaac ttgacgattc caaatcctgt caccgagttg atggataccc
      961 caatcaacag cacgtatttc caaaacctta atgcggaaac ggacggcgga aatcattaca
     1021 tctacaatgc cttcacagcc accgattaca acatttgtgc agcttacctt ttcttcattg
     1081 cttgtctggg agtcagtctc aacgttttgg tgttggtcct ctttatcaaa gacagaaagt
     1141 tgaggagccc caataacttc ctgtatgtaa gtctcgctct cggggatctg ttggttgcag
     1201 ttttcggcac cgcatttaag ttcattatta cagctcgcaa aaccctgttg cgagaagaag
     1261 acggtttctg caaatggtac ggcttcataa catacctcgg cgggctcgcg gcactgatga
     1321 ccctgagcgt tattgctttt gttcgttgtc tggccgtgct gcgtctcggc tccttcacag
     1381 gtttgacaac gcggatgggc gtggcggcga tggctttcat ttggatctac tccctggcat
     1441 tcacactcgc gcctctcctt gggtggaatc actacatacc ggaaggtttg gccacttggt
     1501 gtagtatcga ctggctttcc gatgaaacat ccgataagtc ctacgtgttt gctattttca
     1561 tcttctgctt tcttgtccct gttctcatta tcgtcgtgtc atacgggctg atctacgata
     1621 aggttcggaa ggtagcgaaa acaggcggtt ctgttgccaa agcagaacgc gaagtactgc
     1681 gcatgacgct gctgatggtt agcctgttca tgcttgcctg gagtccctac gctgttatct
     1741 gcatgctcgc tagcttcggc cctaaagacc tgctccatcc agtggccaca gtgattcctg
     1801 ccatgtttgc aaagtcttct acgatgtata accctcttat ctatgtgttc atgaacaaac
     1861 aattcaggcg gagccttaaa gttcttctcg gaatgggagt agaggacctg aactccgaga
     1921 gtgagagagc aactggtggt acagccacga atcaagtcgc cgcgacgacc gagacttccc
     1981 aggttgctcc tgcttaaaag cttaatcaac ctctggatta caaaatttgt gaaagattga
     2041 ctgatattct taactatgtt gctcctttta cgctgtgtgg atatgctgct ttaatgcctc
     2101 tgtatcatgc tattgcttcc cgtatggctt tcgttttctc ctccttgtat aaatcctggt
     2161 tgctgtctct ttatgaggag ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg
     2221 tgtttgctga cgcaaccccc actggctggg gcattgccac cacctgtcaa ctcctttctg
     2281 ggactttcgc tttccccctc ccgatcgcca cggcagaact catcgccgcc tgccttgccc
     2341 gctgctggac aggggctagg ttgctgggca ctgataattc cgtggtgttg tcggggaagc
     2401 tgacgtcagc gctaataaaa tcgctatcca tcgaagatgg atgtgtgttg gttttttgtg
     2461 tgggtaaccc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     2521 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     2581 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     2641 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     2701 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2761 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     2821 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2881 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     2941 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3001 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     3061 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     3121 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     3181 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     3241 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3301 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     3361 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3421 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3481 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3541 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3601 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3661 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3721 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3781 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3841 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3901 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3961 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4021 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4081 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4141 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4201 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4261 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4321 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4381 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4441 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4501 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4561 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4621 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4681 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4741 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4801 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4861 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4921 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4981 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5041 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5101 ggttttgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5161 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5221 ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg
     5281 gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac
     5341 tccatcacta ggggttcctg cggccgcacg cgtcttgtgg actaagtttg ttcgcatccc
     5401 cttctccaac cccctcagta catcaccctg ggggaacagg gtccacttgc tcctgggccc
     5461 acacagtcct gcagtattgt gtatataagg ccagggcaaa gaggagcagg ttttaaagtg
     5521 aaaggcaggc aggtgttggg gaggcagtta ccggggcaac gggaacaggg cgtttcggag
     5581 gtggttgcca tggggacctg gatgctgacg aaggctcgcg aggctgtgag cagccacagt
     5641 gccctgctca gaagccccaa gctcgtcagt caagccggtt ctccgtttgc actcaggagc
     5701 acgggcaggc gagtggcccc tagttctggg ggcagctcta gagcggtacc ggatccatgg
     5761 ctgagctgat cagcagcgcc accagatctc tgtttgccgc cggaggcatc aacccttggc
     5821 ctaaccccta ccaccacgag gacatgggct gtggaggaat gacacctaca ggcgagtgct
     5881 tcagcaccga gtggtggtgt gacccttctt acggactgag cgacgccgga tacggatatt
     5941 gcttcgtgga ggccacaggc ggctacctgg tcgtgggagt ggagaagaag caggcttggc
     6001 tgcacagcag aggcacacca ggagaaaaga tcggcgccca ggtctgccag tggattgctt
     6061 tcagcatcgc catcgccctg ctgacattct acggcttcag cgcctggaag gccacttgcg
     6121 gttgggagga ggtctacgtc tgttgcgtcg aggtgctgtt cgtgaccctg gagatcttca
     6181 aggagttcag cagccccgcc acagtgtacc tgtctaccgg caaccacgcc tattgcctgc
     6241 gctacttcga gtggctgctg tcttgccccg tgatcctgat cagactgagc aacctgagcg
     6301 gcctgaagaa cgactacagc aagcggacca tgggcctgat cgtgtcttgc gtgggaatga
     6361 tcgtgttcgg catggccgca ggactggcta ccgattggct caagtggctg ctgtatatcg
     6421 tgtcttgcat ctacggcggc tacatgtact tccaggccgc caagtgctac gtggaagcca
     6481 accacagcgt gcctaaaggc cattgccgca tggtcgtgaa gctgatggcc tacgcttact
     6541 tcgcctcttg gggcagctac ccaatcctct gggcagtggg accagaagga ctgctgaagc
     6601 tgagccctta cgccaacagc atcggccaca gcatctgcga catcatcgcc aaggagtttt
     6661 ggaccttcct ggcccaccac ctgaggatca agatccacga gcacatcctg atccacggcg
     6721 acatccggaa gaccaccaag atggagatcg gaggcgagga ggtggaagtg gaagagttcg
     6781 tggaggagga ggacgaggac acagtggcgg caccggtagt agcagtgagc aagggcgagg
     6841 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     6901 agttcagcgt gtccg
//
