LOCUS       sequence-466631-        7313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the consitutively active kinase activity 
            recorder positive control Kinprola_on in mammalian cell lines fused 
            to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7313)
  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 7313)
  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_on-mEGFP
FEATURES             Location/Qualifiers
     source          1..7313
                     /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(2513..2515,2516..2518,2519..3232)
                     /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(2558..2579)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2819..2838)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3166..3187
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3264..3281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3270..3494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3627..3646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3778..3825
                     /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             3833..4855
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3833..4855
                     /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    4985..5106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5022..5041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5076..5095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5155..5171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5155..5171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5168..5190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5179..5195
                     /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(5203..5209,5210..5227,5228..5233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5248..5269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5386..5403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5557..6146)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5638..5657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6317..7108,7109..7177))
                     /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     6940..6959
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7178..7282)
                     /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 ggaggttctt ttgcccgcga gaccttccag gccttccgca ccaccgacgt
     1021 cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt
     1081 cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt
     1141 tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa
     1201 catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct
     1261 gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa
     1321 aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga
     1381 caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg agattggtgg
     1441 caccggtggc tctggcggta ccggtggctc tggcggttct atcggtactg gctttccatt
     1501 cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg ttggtccgcg
     1561 cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg tgtggcgcaa
     1621 catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga tcggtatggg
     1681 caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct tcatggatgc
     1741 cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact ggggctccgc
     1801 tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg catttatgga
     1861 gttcatccgc cctatcccga cctgggacga atgggctccg ggttctctgc gtcgcgccac
     1921 cctggtggac cctcctcctc cgccaccgcc accaccgccg ccgccgccac caccacctcc
     1981 tccgcctcct ccaccaccac cgccaccgcc tccaccacct ggcggcagcg cctgtgacta
     2041 tcatttagga aacatttcaa gactgtcaaa taagcatttc caaatactac taggagaaga
     2101 cggtaacctt ttattgaatg acatttccac taatgggacc tggttaaatg ggcaaaaagt
     2161 cgagaagaac agctatcagt tactgtctca aggtgatgaa ataaccgttg gtgtaggcgt
     2221 ggaatcagat attttatctc tggtcatttt cataaacgac aaatttaagc agtgcctcga
     2281 gcagaacaaa gttgatcgcg gtggcggtat gcataagttt tctcaagaac agatcggcga
     2341 aaacattgtg tgcagggtca tttgtaccac gggtcaaatt cccatccgag atttgtcagc
     2401 tgatatttca caagtgctta aggaaaaacg atccataaag aaagtttgga catttggtag
     2461 aaacccaggt tctgccaagg agaccttcca ggccttccgc agcggaggtt ctatggtgag
     2521 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2581 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2641 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2701 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2761 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2821 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2881 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2941 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     3001 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     3061 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3121 cacccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3181 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aatctagagg
     3241 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3301 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3361 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3421 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3481 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3541 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3781 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3841 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3901 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3961 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4021 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     4081 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4141 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     4201 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4261 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4321 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4381 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4441 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4501 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4561 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4621 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4681 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4741 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4801 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4861 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4921 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4981 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5041 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5101 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5161 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5221 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5281 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5341 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5401 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5461 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5521 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gccccccctg
     5581 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5641 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5701 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5761 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5821 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5881 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5941 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6001 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6061 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6121 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6181 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6241 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6301 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6361 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6421 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6481 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6541 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6601 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6661 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccta
     6721 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6781 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6841 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6901 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6961 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7021 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7081 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7141 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7201 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7261 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
