LOCUS       sequence-390653-        6819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optimized PdCO in frame with mScarlet under control of 
            eukaryotic Ef1a promoter after Cre-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-DIO-PdCO-mScarlet-ER-miniWPRE
FEATURES             Location/Qualifiers
     source          1..6819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   43..172
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        273..1451
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          504..1442
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1399..1419
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1482..1515)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1566..1599)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1638..2333)
                     /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(1913..1932)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2182..2200
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3506..3515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3531..3564
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3615..3648
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3729..3749)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4124..4264
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4124..4253
                     /label=AAV2 ITR
     rep_origin      4339..4794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4426..4445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4636..4657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4811..4830)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4930..4952
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4990..5008)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5076..5180
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5181..5249,5250..6041)
                     /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(5399..5418)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6212..6800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6701..6720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg
       61 ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
      121 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc
      181 acgcgtaagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt tgcagctaat
      241 ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc
      301 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
      361 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
      421 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
      481 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
      541 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc
      601 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
      661 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa
      721 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
      781 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
      841 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
      901 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
      961 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
     1021 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     1081 ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     1141 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     1201 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     1261 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga
     1321 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     1381 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc
     1441 aggtgtcgtg aggtaccgga tcctctagag tcgactccgg aataacttcg tataggatac
     1501 tttatacgaa gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc
     1561 tcttaataac ttcgtatagc atacattata cgaagttatg gcgcgccgtc gacttacacc
     1621 tcgttctcgt agcagaactt gtacagctcg tccatgccgc cggtggagtg gcggccctcg
     1681 gagcgttcgt actgttccac cacggtgtag tcctcgttgt gggaggtgat gtccaacttg
     1741 cggtcgacgt tgtaggcgcc gggcatctgc acgggcttct tggccttgta ggtggtcttg
     1801 aagtccgcca ggtagcgtcc gccgtccttc aggcgcaggg ccatcttaat gtcgcccttc
     1861 agcacgccgt cctcggggta caaccgctcg gtggacgctt cccagcccat tgtcttcttc
     1921 tgcattacgg ggccgtcagg agggaagttg gtgccgcgga gcttcacctt gtagatcagg
     1981 gtgccgtcct ccagggaggt gtcctgggtc acggtcacgg cgccgccgtc ctcgaagttc
     2041 atcacgcgct cccacttgaa gccctcgggg aaggactgct tatagtagtc ggggatgtcg
     2101 gcggggtgct tggtgaaggc cctggagccg tacatgaact gaggggacag gatgtcccag
     2161 gagaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag
     2221 gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcatgga gccctccatg
     2281 tgcaccttga accgcatgaa ctccttgatc actgcctcgc ccttgctcac catcaccacg
     2341 ttgatgtcga tctggtccag ggggatgtac tcgccctcgc tggtgatcct gctcttaccg
     2401 gttggatctc gggctcgcgg agcaggagca acctgggaag tctcggtcgt cgcggcgact
     2461 tgattcgtgg ctgtaccacc agttgctctc tcactctcgg agttcaggtc ctctactccc
     2521 attccgagaa gaactttaag gctccgcctg aattgtttgt tcatgaacac atagataaga
     2581 gggttataca tcgtagaaga ctttgcaaac atggcaggaa tcactgtggc cactggatgg
     2641 agcaggtctt tagggccgaa gctggcgagc atgcagataa cagcgtaggg actccaggca
     2701 agcatgaaca ggctaaccat cagcagcgtc atgcgcagta cttcgcgttc tgctttggca
     2761 acagaaccgc ctgttttcgc taccttccga accttatcgt agatcagccc gtatgacacg
     2821 acgataatga gaacagggac aagaaagcag aagatgaaaa tagcaaacac gtaggactta
     2881 tcggatgttt catcggaaag ccagtcgata ctacaccaag tggccaaacc ttccggtatg
     2941 tagtgattcc acccaaggag aggcgcgagt gtgaatgcca gggagtagat ccaaatgaaa
     3001 gccatcgccg ccacgcccat ccgcgttgtc aaacctgtga aggagccgag acgcagcacg
     3061 gccagacaac gaacaaaagc aataacgctc agggtcatca gtgccgcgag cccgccgagg
     3121 tatgttatga agccgtacca tttgcagaaa ccgtcttctt ctcgcaacag ggttttgcga
     3181 gctgtaataa tgaacttaaa tgcggtgccg aaaactgcaa ccaacagatc cccgagagcg
     3241 agacttacat acaggaagtt attggggctc ctcaactttc tgtctttgat aaagaggacc
     3301 aacaccaaaa cgttgagact gactcccaga caagcaatga agaaaaggta agctgcacaa
     3361 atgttgtaat cggtggctgt gaaggcattg tagatgtaat gatttccgcc gtccgtttcc
     3421 gcattaaggt tttggaaata cgtgctgttg attggggtat ccatcaactc ggtgacagga
     3481 tttggaatcg tcaagttctc cccgtccatg gtggcaacag cgctgctagc ataacttcgt
     3541 ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta
     3601 gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc
     3661 aagcttatcg atgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3721 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3781 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     3841 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3901 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     3961 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4021 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4081 gatgtgggag gttttttaaa gcacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     4141 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4201 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4261 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4321 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4381 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4441 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4501 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4561 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4621 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     4681 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     4741 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     4801 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4861 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4921 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4981 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5041 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5101 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5161 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5221 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5281 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5341 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5401 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5461 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5521 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5581 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5641 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5701 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5761 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5821 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5881 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5941 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6001 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6061 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6121 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6181 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6241 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6301 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6361 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6421 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6481 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6541 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6601 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6661 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6721 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6781 gggggcggag cctatggaaa aacgccagca acgcggcct
//
