LOCUS       sequence-351721-        8136 bp    DNA     circular SYN 15-APR-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 8136)
  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 8136)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..8136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          140..1156
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1080..1102
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1164..1183
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1234..1243
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3757..3759,3760..3762,3763..4476)
                     /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(3757..3759,3760..3762,3763..4473)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3802..3823)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4063..4082)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4410..4431
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4495..4515)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4495..4513)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4534..4550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4652..4671)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4717..4772
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4771..4790)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5133..5149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5157..5173
                     /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(5181..5187,5188..5205,5206..5211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5226..5247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5306..5501
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5352..5487
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5507..5641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5544..5563)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5598..5617
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5709..5726)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5880..6468)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5960..5979)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6639..7430,7431..7499))
                     /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     7262..7281
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7500..7604)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        7635..8014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(8016..8136,1..139)
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
ORIGIN
        1 ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
       61 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
      121 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
      181 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
      241 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
      301 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag
      361 cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct
      421 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg
      481 cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa
      541 aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg
      601 gctgcaaccc cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc
      661 ggggctccgt acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt
      721 gggggtgccg ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg
      781 cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg
      841 gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc
      901 tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag
      961 gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct
     1021 ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg
     1081 gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt
     1141 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     1201 caaagaattc atagtagcta gcagttgctc gaggccacca tggcgccgtt cctgcgcatc
     1261 gccttcaact cctatgagct gggctccctg caggccgagg acgaggcgaa ccagcccttc
     1321 tgtgccgtga agatgaagga ggcgctcagc acagagcgtg ggaaaacact ggtgcagaag
     1381 aagccgacca tgtatcctga gtggaagtcg acgttcgatg cccacatcta tgaggggcgc
     1441 gtcatccaga ttgtgctaat gcgggcagca gaggagccag tgtctgaggt gaccgtgggt
     1501 gtgtcggtgc tggccgagcg ctgcaagaag aacaatggca aggctgagtt ctggctggac
     1561 ctgcagcctc aggccaaggt gttgatgtct gttcagtatt tcctggagga cgtggattgc
     1621 aaacagtcta tgcgcagtga ggacgaggcc aagttcccaa cgatgaaccg ccgcggagcc
     1681 atcaaacagg ccaaaatcca ctacatcaag aaccatgagt ttatcgccac cttctttggg
     1741 caacccacct tctgttctgt gtgcaaagac tttgtctggg gcctcaacaa gcaaggctac
     1801 aaatgcaggc aatgtaacgc tgccatccac aagaaatgca tcgacaagat catcggcaga
     1861 tgcactggca ccgcggccaa cagccgggac actatattcc agaaagaacg cttcaacatc
     1921 gacatgccga ccggttctgg gtccgcagcc agtagcgccg gaagtatgat cagtctgatt
     1981 gcggcgttag cggtagatcg cgttatcggc atggaaaacg ccatgccgtg gaacctgcct
     2041 gccgatctcg cctggtttaa acgcaacacc ttaaataaac ccgtgattat gggccgccat
     2101 acctgggaat caatcggtcg tccgttgcca ggacgcaaaa atattatcct cagcagtcaa
     2161 ccgggtacgg acaaaaaaaa gaaaaagaaa gatcgcgtaa cgtgggtgaa gtcggtggat
     2221 gaagccatcg cggcgtgtgg tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt
     2281 tatgaacagt tcttgccaaa agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg
     2341 gaaggcgaca cccatttccc ggattacgag ccggatgact gggaatcggt attcagcgaa
     2401 ttccacgatg ctgatgcgca gaactctcac agctattgct ttgagattct ggagcggcgg
     2461 ggtgcttcgg gtggcggcgg atcttcagct cccgggggca tcaaccagaa gcttttggct
     2521 gaggccttga accaagtcac ccagagagcc tcccggagat cagactcagc ctcctcagag
     2581 cctgttggga tatatcaggg tttcgagaag aagaccggag ttgctgggga ggacatgcaa
     2641 gacaacagtg ggacctacgg caagatctgg gagggcagca gcaagtgcaa catcaacaac
     2701 ttcatcttcc acaaggtcct gggcaaaggc agcttcggga aggtgctgct tggagagctg
     2761 aagggcagag gagagtactt tgccatcaag gccctcaaga aggatgtggt cctgatcgac
     2821 gacgacgtgg agtgcaccat ggttgagaag cgggtgctga cacttgccgc agagaatccc
     2881 tttctcaccc acctcatctg caccttccag accaaggacc acctgttctt tgtgatggag
     2941 ttcctcaacg ggggggacct gatgtaccac atccaggaca aaggccgctt tgaactctac
     3001 cgtgccacgt tttatgccgc tgagataatg tgtggactgc agtttctaca cagcaagggc
     3061 atcatttaca gggacctcaa actggacaat gtgctgttgg accgggatgg ccacatcaag
     3121 attgccgact ttgggatgtg caaagagaac atattcgggg agagccgggc cagcaccttc
     3181 tgcggcaccc ctgactatat cgcccctgag atcctacagg gcctgaagta cacattctct
     3241 gtggactggt ggtctttcgg ggtccttctg tacgagatgc tcattggcca gtcccccttc
     3301 catggtgatg atgaggatga actcttcgag tccatccgtg tggacacgcc acattatccc
     3361 cgctggatca ccaaggagtc caaggacatc ctggagaagc tctttgaaag ggaaccaacc
     3421 aagaggctgg gagtgacggg aaacatcaaa atccacccct tcttcaagac cataaactgg
     3481 actctgctgg aaaagcggag gttggagcca cccttcaggc ccaaagtgaa gtcacccaga
     3541 gactacagta actttgacca ggagttcctg aacgagaagg cgcgcctctc ctacagcgac
     3601 aagaacctca tcgactccat ggaccagtct gcattcgctg gcttctcctt tgtgaacccc
     3661 aaattcgagc acctcctgga agatgcggcc gctggtagtg gtgctggctc tggtgctggt
     3721 agtggcgctg gttccggtgc tggctcttcg gccgctatgg tgagcaaggg cgaggagctg
     3781 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3841 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3901 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3961 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     4021 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     4081 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     4141 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     4201 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4261 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4321 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     4381 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     4441 gggatcactc tcggcatgga cgagctgtac aagtaggagc tccagctttt gttcccttta
     4501 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4561 ttatccgcta attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     4621 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     4681 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     4741 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     4801 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     4861 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
     4921 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
     4981 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
     5041 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc
     5101 tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5161 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5221 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5281 ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc
     5341 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5401 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     5461 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc
     5521 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5581 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     5641 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5701 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5761 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5821 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5881 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5941 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6001 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6061 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6121 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6181 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6241 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6301 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6361 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6421 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6481 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6541 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6601 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6661 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6721 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6781 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6841 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6901 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6961 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7021 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7081 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7141 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7201 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7261 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7321 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7381 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7441 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7501 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7561 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7621 gtgccacctg ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     7681 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     7741 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     7801 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     7861 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     7921 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     7981 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
     8041 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
     8101 attttgtgca gcgatggggg cggggggggg gggggg
//
