LOCUS       sequence-390702-        7464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optimized PdCO in frame with EGFP under control of CMV 
            promotor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7464)
  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 7464)
  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: pcDNA3.1-CMV-PdCO-EGFP
FEATURES             Location/Qualifiers
     source          1..7464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(71..90)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        262..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     890..909
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        890..908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      944..953
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2126..2128,2129..2131,2132..2842)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2126..2128,2129..2131,2132..2842)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2171..2192)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2432..2451)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2779..2800
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2913..2930)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2919..3143
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3189..3617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3276..3295)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3486..3507
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3626..3646)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3631..3960
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3811..3946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3873..3892
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4009..5034
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4009..5034
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5164..5285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5201..5220)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5255..5274
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5334..5350)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5334..5350)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5347..5369)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5358..5374
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5382..5388,5389..5406,5407..5412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5427..5448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5565..5582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5736..6324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5816..5835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6495..7286,7287..7355))
                     /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     7118..7137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7356..7460)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
       61 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      121 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      181 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      241 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      781 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      901 actataggga gacccaagct ggctagcgtt taaacttgga tccgccacca tggacgggga
      961 gaacttgacg attccaaatc ctgtcaccga gttgatggat accccaatca acagcacgta
     1021 tttccaaaac cttaatgcgg aaacggacgg cggaaatcat tacatctaca atgccttcac
     1081 agccaccgat tacaacattt gtgcagctta ccttttcttc attgcttgtc tgggagtcag
     1141 tctcaacgtt ttggtgttgg tcctctttat caaagacaga aagttgagga gccccaataa
     1201 cttcctgtat gtaagtctcg ctctcgggga tctgttggtt gcagttttcg gcaccgcatt
     1261 taagttcatt attacagctc gcaaaaccct gttgcgagaa gaagacggtt tctgcaaatg
     1321 gtacggcttc ataacatacc tcggcgggct cgcggcactg atgaccctga gcgttattgc
     1381 ttttgttcgt tgtctggccg tgctgcgtct cggctccttc acaggtttga caacgcggat
     1441 gggcgtggcg gcgatggctt tcatttggat ctactccctg gcattcacac tcgcgcctct
     1501 ccttgggtgg aatcactaca taccggaagg tttggccact tggtgtagta tcgactggct
     1561 ttccgatgaa acatccgata agtcctacgt gtttgctatt ttcatcttct gctttcttgt
     1621 ccctgttctc attatcgtcg tgtcatacgg gctgatctac gataaggttc ggaaggtagc
     1681 gaaaacaggc ggttctgttg ccaaagcaga acgcgaagta ctgcgcatga cgctgctgat
     1741 ggttagcctg ttcatgcttg cctggagtcc ctacgctgtt atctgcatgc tcgctagctt
     1801 cggccctaaa gacctgctcc atccagtggc cacagtgatt cctgccatgt ttgcaaagtc
     1861 ttctacgatg tataaccctc ttatctatgt gttcatgaac aaacaattca ggcggagcct
     1921 taaagttctt ctcggaatgg gagtagagga cctgaactcc gagagtgaga gagcaactgg
     1981 tggtacagcc acgaatcaag tcgccgcgac gaccgagact tcccaggttg ctcctgctcc
     2041 gcgagcccga gatccaaccg gtaagagcag gatcaccagc gagggcgagt acatccccct
     2101 ggaccagatc gacatcaacg tggtgatggt gagcaagggc gaggagctgt tcaccggggt
     2161 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2221 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcattt gcaccaccgg
     2281 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2341 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2401 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2461 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2521 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2581 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2641 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2701 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2761 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2821 cggcatggac gagctgtaca agttctgcta cgagaacgag gtgtaactag aattcaagct
     2881 ttctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2941 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3001 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3061 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3121 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3181 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3241 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3301 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3361 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3421 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3481 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatcttgg tctattcttt
     3541 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3601 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3661 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3721 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3781 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3841 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3901 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3961 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgat gaaaaagcct
     4021 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     4081 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     4141 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     4201 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     4261 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     4321 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     4381 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     4441 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4501 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4561 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4621 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4681 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4741 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4801 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4861 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4921 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4981 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgtg
     5041 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     5101 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     5161 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5221 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5281 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5341 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5401 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5461 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5581 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5641 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5701 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5761 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5821 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5881 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5941 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6001 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6061 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6121 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6181 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6241 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6301 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6361 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6421 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6481 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6541 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6601 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6661 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6721 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6781 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6841 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6901 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6961 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7021 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7081 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7141 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7201 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7261 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7321 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7381 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7441 aaacaaatag gggttccgcg caca
//
