LOCUS       sequence-398292-        6819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optimized PdCO in frame with mScarlet under control of 
            eukaryotic Ef1a promoter after Flp-dependent recombination..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6819)
  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 6819)
  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_EF1a-fDIO-PdCO-mScarlet-miniWPRE
FEATURES             Location/Qualifiers
     source          1..6819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..135
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        236..1414
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          467..1405
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1362..1382
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1445..1478
                     /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(1601..2296)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1876..1895)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2145..2163
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3469..3478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3494..3527)
                     /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)"
     primer_bind     complement(3692..3712)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4087..4227
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4087..4216
                     /label=AAV2 ITR
     rep_origin      4302..4757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4389..4408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4599..4620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4774..4793)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4893..4915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4953..4971)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5039..5143
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5144..5212,5213..6004)
                     /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(5362..5381)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6175..6763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6664..6683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
       61 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      121 tcactagggg ttcctgcggc cgcacgcgta agctttgcaa agatggataa agttttaaac
      181 agagaggaat ctttgcagct aatggacctt ctaggtcttg aaaggagtgg gaattggctc
      241 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      301 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      361 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      421 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
      481 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
      541 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
      601 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
      661 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
      721 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
      781 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
      841 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
      901 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
      961 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     1021 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     1081 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     1141 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     1201 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     1261 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     1321 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     1381 tcaaagtttt tttcttccat ttcaggtgtc gtgaggtacc ggatcctcta gagtcgactc
     1441 cggagaagtt cctattctct agaaagtata ggaacttcgc agaatggtag ctggattgta
     1501 gctgctatta gcaatatgaa acctcttaga agttcctatt cttcaaaagg tataggaact
     1561 tcggcgcgcc gtcgacttac acctcgttct cgtagcagaa cttgtacagc tcgtccatgc
     1621 cgccggtgga gtggcggccc tcggagcgtt cgtactgttc caccacggtg tagtcctcgt
     1681 tgtgggaggt gatgtccaac ttgcggtcga cgttgtaggc gccgggcatc tgcacgggct
     1741 tcttggcctt gtaggtggtc ttgaagtccg ccaggtagcg tccgccgtcc ttcaggcgca
     1801 gggccatctt aatgtcgccc ttcagcacgc cgtcctcggg gtacaaccgc tcggtggacg
     1861 cttcccagcc cattgtcttc ttctgcatta cggggccgtc aggagggaag ttggtgccgc
     1921 ggagcttcac cttgtagatc agggtgccgt cctccaggga ggtgtcctgg gtcacggtca
     1981 cggcgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggact
     2041 gcttatagta gtcggggatg tcggcggggt gcttggtgaa ggccctggag ccgtacatga
     2101 actgagggga caggatgtcc caggagaagg gcagggggcc acccttggtc accttcagct
     2161 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt
     2221 ggccgttcat ggagccctcc atgtgcacct tgaaccgcat gaactccttg atcactgcct
     2281 cgcccttgct caccatcacc acgttgatgt cgatctggtc cagggggatg tactcgccct
     2341 cgctggtgat cctgctctta ccggttggat ctcgggctcg cggagcagga gcaacctggg
     2401 aagtctcggt cgtcgcggcg acttgattcg tggctgtacc accagttgct ctctcactct
     2461 cggagttcag gtcctctact cccattccga gaagaacttt aaggctccgc ctgaattgtt
     2521 tgttcatgaa cacatagata agagggttat acatcgtaga agactttgca aacatggcag
     2581 gaatcactgt ggccactgga tggagcaggt ctttagggcc gaagctggcg agcatgcaga
     2641 taacagcgta gggactccag gcaagcatga acaggctaac catcagcagc gtcatgcgca
     2701 gtacttcgcg ttctgctttg gcaacagaac cgcctgtttt cgctaccttc cgaaccttat
     2761 cgtagatcag cccgtatgac acgacgataa tgagaacagg gacaagaaag cagaagatga
     2821 aaatagcaaa cacgtaggac ttatcggatg tttcatcgga aagccagtcg atactacacc
     2881 aagtggccaa accttccggt atgtagtgat tccacccaag gagaggcgcg agtgtgaatg
     2941 ccagggagta gatccaaatg aaagccatcg ccgccacgcc catccgcgtt gtcaaacctg
     3001 tgaaggagcc gagacgcagc acggccagac aacgaacaaa agcaataacg ctcagggtca
     3061 tcagtgccgc gagcccgccg aggtatgtta tgaagccgta ccatttgcag aaaccgtctt
     3121 cttctcgcaa cagggttttg cgagctgtaa taatgaactt aaatgcggtg ccgaaaactg
     3181 caaccaacag atccccgaga gcgagactta catacaggaa gttattgggg ctcctcaact
     3241 ttctgtcttt gataaagagg accaacacca aaacgttgag actgactccc agacaagcaa
     3301 tgaagaaaag gtaagctgca caaatgttgt aatcggtggc tgtgaaggca ttgtagatgt
     3361 aatgatttcc gccgtccgtt tccgcattaa ggttttggaa atacgtgctg ttgattgggg
     3421 tatccatcaa ctcggtgaca ggatttggaa tcgtcaagtt ctccccgtcc atggtggcaa
     3481 cagcgctgct agcgaagttc ctatactttc tagagaatag gaacttcttg ccttaaccca
     3541 gaaattatca ctgttattct ttagaatggt gcaaagagaa gttcctatac cttttgaaga
     3601 ataggaactt cgaattcgat atcaagctta tcgatgataa tcaacctctg gattacaaaa
     3661 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3721 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3781 tgtataaatc ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct
     3841 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt
     3901 gtgatgctat tgctttattt gtaaccatct agctttattt gtgaaatttg tgatgctatt
     3961 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4021 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcacgtg cggaccgagc
     4081 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4141 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4201 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4261 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4321 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     4381 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4441 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4501 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4561 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4621 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4681 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4741 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4801 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4861 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4921 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4981 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5041 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5101 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5161 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5221 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5281 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5341 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5401 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5461 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5521 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5581 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5641 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5701 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5761 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5821 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5881 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5941 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6001 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6061 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6121 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6181 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6241 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6301 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6361 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6421 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6481 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6541 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6601 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6661 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6721 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6781 ctttttacgg ttcctggcct tttgctggcc ttttgctca
//
