LOCUS       sequence-466619-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the PKC activity recorder Kinprola_PKC in 
            mammalian cell lines fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  AUTHORS   Sun DE, Ng SW, Zheng Y, Xie S, Schwan N, Breuer P, Hoffmann DC, 
            Michel J, Azorin DD, Boonekamp KE, Winkler F, Wick W, Boutros M, Li 
            Y, Johnsson K
  TITLE     Molecular recording of cellular protein kinase activity with 
            chemical labeling.
  JOURNAL   Nat Chem Biol. 2025 Jul 10. doi: 10.1038/s41589-025-01949-6.
  PUBMED    40640555
REFERENCE   2  (bases 1 to 7295)
  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 Chem 
            Biol. 2025 Jul 10. doi: 10.1038/s41589-025-01949-6."
COMMENT     Sequence Label: pCDNA5/FRT_CMV_NES-Kinprola_PKC-mEGFP
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2495..2497,2498..2500,2501..3214)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2540..2561)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2801..2820)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3148..3169
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3246..3263)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3252..3476
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3609..3628)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3760..3807
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3815..4837
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3815..4837
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4967..5088
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5004..5023)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5058..5077
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5137..5153)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5137..5153)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5150..5172)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5161..5177
                     /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(5185..5191,5192..5209,5210..5215))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5230..5251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5368..5385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5539..6128)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5620..5639)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6299..7090,7091..7159))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNIGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6922..6941
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7160..7264)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt accaccatgc tgcagaacga gcttgctctt aagttggctg gacttgatat
      961 taacaagact ggaggttctg acgtcggccg caagctgatc atcgatcaga acgtttttat
     1021 cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta
     1081 ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct
     1141 gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct
     1201 gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc
     1261 ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc
     1321 gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg
     1381 gctgtctact ctggagattg gtggcaccgg tggctctggc ggtaccggtg gctctggcgg
     1441 ttctatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
     1501 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
     1561 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     1621 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     1681 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     1741 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     1801 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatgggc
     1861 tccgggttct agattcagac gctttcagac tctcaaagac aaggctaagg ctcctcctcc
     1921 tccgccaccg ccaccaccgc cgccgccgcc accaccacct cctccgcctc ctccaccacc
     1981 accgccaccg cctccaccac ctggcggcag cgcctgtgac tatcatttag gaaacatttc
     2041 aagactgtca aataagcatt tccaaatact actaggagaa gacggtaacc ttttattgaa
     2101 tgacatttcc actaatggga cctggttaaa tgggcaaaaa gtcgagaaga acagctatca
     2161 gttactgtct caaggtgatg aaataaccgt tggtgtaggc gtggaatcag atattttatc
     2221 tctggtcatt ttcataaacg acaaatttaa gcagtgcctc gagcagaaca aagttgatcg
     2281 cggtggcggt atgcataagt tttctcaaga acagatcggc gaaaacattg tgtgcagggt
     2341 catttgtacc acgggtcaaa ttcccatccg agatttgtca gctgatattt cacaagtgct
     2401 taaggaaaaa cgatccataa agaaagtttg gacatttggt agaaacccag gttctgccaa
     2461 ggagaccttc caggccttcc gcagcggagg ttctatggtg agcaagggcg aggagctgtt
     2521 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2581 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2641 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2701 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2761 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2821 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2881 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2941 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3001 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3061 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaaactgag
     3121 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3181 gatcactctc ggcatggacg agctgtacaa gtaatctaga gggcccgttt aaacccgctg
     3241 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3301 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3361 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3421 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3481 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3541 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3601 agcgcccgct cctttcgctt tcttcccttc ctttctagcc acgttcgccg gctttccccg
     3661 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3721 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     3781 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     3841 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     3901 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     3961 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     4021 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     4081 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     4141 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     4201 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     4261 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacacagt
     4321 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     4381 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     4441 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     4501 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     4561 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     4621 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     4681 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     4741 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     4801 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     4861 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4921 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4981 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5041 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     5101 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5161 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5221 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5281 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5341 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5401 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5461 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5521 ggccgcgttg ctggcgtttt tccataggct ccgccccccc tgacgagcat cacaaaaatc
     5581 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5641 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5701 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5761 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5821 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5881 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5941 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6001 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6061 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6121 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6181 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6241 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6301 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6361 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6421 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6481 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6541 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6601 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6661 gctccggttc ccaacgatca aggcgagtta catgatcccc tatgttgtgc aaaaaagcgg
     6721 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6781 tggttatggc agcactgcat aattctctta ctgtcatacc atccgtaaga tgcttttctg
     6841 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6901 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6961 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7021 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7081 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7141 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7201 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7261 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
