LOCUS       sequence-466630-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV driven expression of the inactive AMPK activity recorder 
            negative control with T/A mutation Kinprola_AMPK_T/A in mammalian 
            cell lines fused to mEGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  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 7283)
  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_AMPK_T/A-mEGFP
FEATURES             Location/Qualifiers
     source          1..7283
                     /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(2483..2485,2486..2488,2489..3202)
                     /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(2528..2549)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2789..2808)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3136..3157
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3234..3251)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3240..3464
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3597..3616)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3748..3795
                     /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             3803..4825
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3803..4825
                     /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    4955..5076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4992..5011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5046..5065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5125..5141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5125..5141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5138..5160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5149..5165
                     /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(5173..5179,5180..5197,5198..5203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5218..5239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5356..5373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5527..6116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5608..5627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6287..7078,7079..7147))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVAPIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNIGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6910..6929
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7148..7252)
                     /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 atgcgtagag tagcggcgct ggtagattta cctcctcctc cgccaccgcc
     1921 accaccgccg ccgccgccac caccacctcc tccgcctcct ccaccaccac cgccaccgcc
     1981 tccaccacct ggcggcagcg cctgtgacta tcatttagga aacatttcaa gactgtcaaa
     2041 taagcatttc caaatactac taggagaaga cggtaacctt ttattgaatg acatttccac
     2101 taatgggacc tggttaaatg ggcaaaaagt cgagaagaac agctatcagt tactgtctca
     2161 aggtgatgaa ataaccgttg gtgtaggcgt ggaatcagat attttatctc tggtcatttt
     2221 cataaacgac aaatttaagc agtgcctcga gcagaacaaa gttgatcgcg gtggcggtat
     2281 gcataagttt tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac
     2341 gggtcaaatt cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg
     2401 atccataaag aaagtttgga catttggtag aaacccaggt tctgccaagg agaccttcca
     2461 ggccttccgc agcggaggtt ctatggtgag caagggcgag gagctgttca ccggggtggt
     2521 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2581 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2641 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2701 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2761 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2821 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2881 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     2941 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3001 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3061 cgtgctgctg cccgacaacc actacctgag cacccagtcc aaactgagca aagaccccaa
     3121 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3181 catggacgag ctgtacaagt aatctagagg gcccgtttaa acccgctgat cagcctcgac
     3241 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3301 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3361 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3421 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3481 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3541 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3601 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttcccagtc aagctctaaa
     3661 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3721 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     3781 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     3841 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     3901 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     3961 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     4021 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     4081 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     4141 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     4201 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     4261 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     4321 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     4381 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     4441 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     4501 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     4561 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     4621 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     4681 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     4741 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     4801 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     4861 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4921 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4981 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5041 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     5101 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5161 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     5221 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5281 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5341 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5401 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5461 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5521 ggcgtttttc cataggctcc gccccccctg acgagcatca caaaaatcga cgctcaagtc
     5581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5941 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6481 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6541 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6601 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6661 aacgatcaag gcgagttaca tgatccccta tgttgtgcaa aaaagcggtt agctccttcg
     6721 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6781 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6841 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6901 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6961 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7021 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7081 caaaaacagg aaggcaaaat gccgcaaaaa agggaatagg ggcgacacgg aaatgttgaa
     7141 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7201 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7261 cccgaaaagt gccacctgac gtc
//
