LOCUS       sequence-466620-        7835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the PKA activity recorder Kinprola_PKA in 
            mammalian cell lines fused to mEGFP, targeting to the microtubule.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7835)
  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 7835)
  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_Kinprola_PKA-mEGFP-MTBD
FEATURES             Location/Qualifiers
     source          1..7835
                     /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(2417..2419,2420..2422,2423..3133)
                     /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(2462..2483)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2723..2742)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3070..3091
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3786..3803)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3792..4016
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4149..4168)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4300..4347
                     /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             4355..5377
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4355..5377
                     /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    5507..5628
                     /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(5677..5693)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5677..5693)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5690..5712)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5701..5717
                     /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(5725..5731,5732..5749,5750..5755))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5770..5791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5908..5925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6079..6668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6160..6179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6839..7630,7631..7699))
                     /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     7462..7481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7700..7804)
                     /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 accaccatgg acgtcggccg caagctgatc atcgatcaga acgtttttat
      961 cgagggtacg ctgccgatgg gtgtcgtccg cccgctgact gaagtcgaga tggaccatta
     1021 ccgcgagccg ttcctgaatc ctgttgaccg cgagccactg tggcgcttcc caaacgagct
     1081 gccaatcgcc ggtgagccag cgaacatcgt cgcgctggtc gaagaataca tggactggct
     1141 gcaccagtcc cctgtcccga agctgctgtt ctggggcacc ccaggcgttc tgatcccacc
     1201 ggccgaagcc gctcgcctgg ccaaaagcct gcctaactgc aaggctgtgg acatcggccc
     1261 gggtctgaat ctgctgcaag aagacaaccc ggacctgatc ggcagcgaga tcgcgcgctg
     1321 gctgtctact ctggagattg gtggcaccgg tggctctggc ggtaccggtg gctctggcgg
     1381 ttctatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat
     1441 gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc
     1501 gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat
     1561 tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt atttcttcga
     1621 cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag aggtcgtcct
     1681 ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca atccagagcg
     1741 cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg acgaatgggc
     1801 tccgggttct ctgcgtcgcg ccaccctggt ggaccctcct cctccgccac cgccaccacc
     1861 gccgccgccg ccaccaccac ctcctccgcc tcctccacca ccaccgccac cgcctccacc
     1921 acctggcggc agcgcctgtg actatcattt aggaaacatt tcaagactgt caaataagca
     1981 tttccaaata ctactaggag aagacggtaa ccttttattg aatgacattt ccactaatgg
     2041 gacctggtta aatgggcaaa aagtcgagaa gaacagctat cagttactgt ctcaaggtga
     2101 tgaaataacc gttggtgtag gcgtggaatc agatatttta tctctggtca ttttcataaa
     2161 cgacaaattt aagcagtgcc tcgagcagaa caaagttgat cgcggtggcg gtatgcataa
     2221 gttttctcaa gaacagatcg gcgaaaacat tgtgtgcagg gtcatttgta ccacgggtca
     2281 aattcccatc cgagatttgt cagctgatat ttcacaagtg cttaaggaaa aacgatccat
     2341 aaagaaagtt tggacatttg gtagaaaccc aggttctgcc aaggagacct tccaggcctt
     2401 ccgcagcgga ggttctatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2461 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2521 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2581 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2641 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2701 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2761 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2821 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2881 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2941 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3001 gctgcccgac aaccactacc tgagcaccca gtccaaactg agcaaagacc ccaacgagaa
     3061 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3121 cgagctgtac aagtccggag tgtacaaggg taccccagga acccccaagt ctgccatctt
     3181 ggtgccgagt gagaagaagg tcgccatcat acgtactcct ccaaaatctc ctgcgactcc
     3241 caagcagctt cggcttatta accaaccact gccagacctg aagaatgtca aatccaaaat
     3301 cggatcaaca gacaacatca aataccagcc taaagggggg caggtacaaa ttgttaccaa
     3361 gaaaatagac ctaagccatg tgacatccaa atgtggctct ctgaagaaca tccgccacag
     3421 gccaggtggc ggacgtgtga aaattgagag tgtaaaacta gatttcaaag aaaaggccca
     3481 agctaaagtt ggttctcttg ataatgctca tcatgtacct ggaggtggta atgtcaagat
     3541 tgacagccaa aagttgaact tcagagagca tgctaaagcc cgtgtggacc atggggctga
     3601 gatcattaca cagtccccag gcagatccag cgtggcatca ccccgacgac tcagcaatgt
     3661 ctcctcgtct ggaagcatca acctgctcga atctcctcag cttgccactt tggctgagga
     3721 tgtcactgct gcactcgcta agcagggctt gtaatctaga gggcccgttt aaacccgctg
     3781 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3841 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3901 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3961 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     4021 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     4081 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4141 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4201 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4261 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     4321 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     4381 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     4441 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     4501 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     4561 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     4621 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     4681 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     4741 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     4801 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     4861 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     4921 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     4981 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     5041 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     5101 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     5161 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     5221 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     5281 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     5341 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     5401 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5461 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     5521 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5581 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     5641 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5701 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5761 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5881 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5941 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6001 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6061 ggccgcgttg ctggcgtttt tccataggct ccgccccccc tgacgagcat cacaaaaatc
     6121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6961 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7201 gctccggttc ccaacgatca aggcgagtta catgatcccc tatgttgtgc aaaaaagcgg
     7261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7801 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
