LOCUS       sequence-347294-        8144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of PKC-delta-eDHFR(69K6) chimera fused to EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8144)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 8144)
  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"; doi: 
            "10.1016/j.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pCAGGS-chPKCdelta/eDHFR(69K6)-EGFP
FEATURES             Location/Qualifiers
     source          1..8144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1748..1757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4271..4273,4274..4276,4277..4990)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4271..4273,4274..4276,4277..4987)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4316..4337)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4577..4596)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4924..4945
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5009..5029)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5009..5027)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5048..5064)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5166..5185)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5231..5286
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5285..5304)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5647..5663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5671..5687
                     /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(5695..5701,5702..5719,5720..5725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5761
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5820..6015
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5866..6001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6021..6155
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6058..6077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6112..6131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6223..6240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6394..6982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6474..6493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7153..7944,7945..8013))
                     /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     7776..7795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8014..8118)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatagta gctagcagtt
     1741 gctcgaggcc accatggcgc cgttcctgcg catcgccttc aactcctatg agctgggctc
     1801 cctgcaggcc gaggacgagg cgaaccagcc cttctgtgcc gtgaagatga aggaggcgct
     1861 cagcacagag cgtgggaaaa cactggtgca gaagaagccg accatgtatc ctgagtggaa
     1921 gtcgacgttc gacgcccaca tctatgaggg gcgcgtcatc cagattgtgc taatgcgggc
     1981 agcagaggag ccagtgtctg aggtgaccgt gggtgtgtcg gtgctggccg agcgctgcaa
     2041 gaagaacaat ggcaaggctg agttctggct ggacctgcag cctcaggcca aggtgttgat
     2101 gtctgttcag tatttcctgg aggacgtgga ttgcaaacag tctatgcgca gtgaggacga
     2161 ggccaagttc ccaacgatga accgccgcgg agccatcaaa caggccaaaa tccactacat
     2221 caagaaccat gagtttatcg ccaccttctt tgggcaaccc accttctgtt ctgtgtgcaa
     2281 agactttgtc tggggcctca acaagcaagg ctacaaatgc aggcaatgta acgctgccat
     2341 ccacaagaaa tgcatcgaca agatcatcgg cagatgcact ggcaccgcgg ccaacagccg
     2401 ggacactata ttccagaaag aacgcttcaa catcgacatg ccgaccggtt ctgggtccgc
     2461 agccagtagc gccggaagta tgatcagtct gattgcggcg ttagcggtag atcgcgttat
     2521 cggcatggaa aacgccatgc cgtggaacct gcctgccgat ctcgcctggt ttaaacgcaa
     2581 caccttaaat aaacccgtga ttatgggccg ccatacctgg gaatcaatcg gtcgtccgtt
     2641 gccaggacgc aaaaatatta tcctcagcag tcaaccgggt acggacaaaa aaaagaaaaa
     2701 gaaagatcgc gtaacgtggg tgaagtcggt ggatgaagcc atcgcggcgt gtggtgacgt
     2761 accagaaatc atggtgattg gcggcggtcg cgtttatgaa cagttcttgc caaaagcgca
     2821 aaaactgtat ctgacgcata tcgacgcaga agtggaaggc gacacccatt tcccggatta
     2881 cgagccggat gactgggaat cggtattcag cgaattccac gatgctgatg cgcagaactc
     2941 tcacagctat tgctttgaga ttctggagcg gcggggtgct tcgggtggcg gcggatcttc
     3001 agctcccggg ggcatcaacc agaagctttt ggctgaggcc ttgaaccaag tcacccagag
     3061 agcctcccgg agatcagact cagcctcctc agagcctgtt gggatatatc agggtttcga
     3121 gaagaagacc ggagttgctg gggaggacat gcaagacaac agtgggacct acggcaagat
     3181 ctgggagggc agcagcaagt gcaacatcaa caacttcatc ttccacaagg tcctgggcaa
     3241 aggcagcttc gggaaggtgc tgcttggaga gctgaagggc agaggagagt actttgccat
     3301 caaggccctc aagaaggatg tggtcctgat cgacgacgac gtggagtgca ccatggttga
     3361 gaagcgggtg ctgacacttg ccgcagagaa tccctttctc acccacctca tctgcacctt
     3421 ccagaccaag gaccacctgt tctttgtgat ggagttcctc aacggggggg acctgatgta
     3481 ccacatccag gacaaaggcc gctttgaact ctaccgtgcc acgttttatg ccgctgagat
     3541 aatgtgtgga ctgcagtttc tacacagcaa gggcatcatt tacagggacc tcaaactgga
     3601 caatgtgctg ttggaccggg atggccacat caagattgcc gactttggga tgtgcaaaga
     3661 gaacatattc ggggagagcc gggccagcac cttctgcggc acccctgact atatcgcccc
     3721 tgagatccta cagggcctga agtacacatt ctctgtggac tggtggtctt tcggggtcct
     3781 tctgtacgag atgctcattg gccagtcccc cttccatggt gatgatgagg atgaactctt
     3841 cgagtccatc cgtgtggaca cgccacatta tccccgctgg atcaccaagg agtccaagga
     3901 catcctggag aagctctttg aaagggaacc aaccaagagg ctgggagtga cgggaaacat
     3961 caaaatccac cccttcttca agaccataaa ctggactctg ctggaaaagc ggaggttgga
     4021 gccacccttc aggcccaaag tgaagtcacc cagagactac agtaactttg accaggagtt
     4081 cctgaacgag aaggcgcgcc tctcctacag cgacaagaac ctcatcgact ccatggacca
     4141 gtctgcattc gctggcttct cctttgtgaa ccccaaattc gagcacctcc tggaagatgc
     4201 ggccgctggt agtggtgctg gctctggtgc tggtagtggc gctggttccg gtgctggctc
     4261 ttcggccgct atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     4321 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     4381 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     4441 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     4501 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     4561 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     4621 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     4681 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     4741 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     4801 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4861 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     4921 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     4981 gtacaagtag gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     5041 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctaattcac tcctcaggtg
     5101 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     5161 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     5221 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     5281 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     5341 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     5401 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     5461 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     5521 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     5581 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     5641 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5701 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     5821 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     5881 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5941 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     6001 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     6061 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     6121 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     6181 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6241 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6301 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6361 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6421 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6481 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6541 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     6601 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6661 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6721 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6781 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     6841 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6901 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6961 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7021 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7081 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7141 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7201 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7261 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7321 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7381 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7441 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7501 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7561 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7621 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7681 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7741 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7801 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7861 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7921 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7981 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8041 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8101 acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
