LOCUS       sequence-466653-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG driven expression of the PKA activity recorder Kinprola_PKA 
            fused to mTagBFP2 for mammalian cell expression through AAV 
            transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  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 6866)
  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: pAAV_CAG_NES-Kinprola_PKA-mTagBFP2_WPRE-SV40
FEATURES             Location/Qualifiers
     source          1..6866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1498..2208
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    2224..2812
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2277..2297)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2695..2706)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2815..2831)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2816..2832)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    2861..2982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2872..2891)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2926..2945
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3144..3273
                     /label=AAV2 ITR
     rep_origin      complement(3510..4098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3590..3609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4269..5060,5061..5129))
                     /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     4892..4911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5130..5234)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5260..5715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5397..5418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5609..5628
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5787..5916
                     /label=AAV2 ITR
     enhancer        5998..6283
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        6285..6558
                     /label=chicken beta-actin promoter
     intron          6559..6786
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 gacgtcggcc gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg
       61 ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat
      121 cctgttgacc gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca
      181 gcgaacatcg tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg
      241 aagctgctgt tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg
      301 gccaaaagcc tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa
      361 gaagacaacc cggacctgat cggcagcgag atcgcgcgct ggctgtctac tctggagatt
      421 ggtggcaccg gtggctctgg cggtaccggt ggctctggcg gttctatcgg tactggcttt
      481 ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt
      541 ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg
      601 cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt
      661 atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg
      721 gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc
      781 tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt
      841 atggagttca tccgccctat cccgacctgg gacgaatggg ctccgggttc tctgcgtcgc
      901 gccaccctgg tggaccctcc tcctccgcca ccgccaccac cgccgccgcc gccaccacca
      961 cctcctccgc ctcctccacc accaccgcca ccgcctccac cacctggcgg cagcgcctgt
     1021 gactatcatt taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga
     1081 gaagacggta accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa
     1141 aaagtcgaga agaacagcta tcagttactg tctcaaggtg atgaaataac cgttggtgta
     1201 ggcgtggaat cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc
     1261 ctcgagcaga acaaagttga tcgcggtggc ggtatgcata agttttctca agaacagatc
     1321 ggcgaaaaca ttgtgtgcag ggtcatttgt accacgggtc aaattcccat ccgagatttg
     1381 tcagctgata tttcacaagt gcttaaggaa aaacgatcca taaagaaagt ttggacattt
     1441 ggtagaaacc caggttctgc caaggagacc ttccaggcct tccgcagcgg aggttctatg
     1501 gtgtctaagg gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc
     1561 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     1621 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     1681 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     1741 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     1801 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     1861 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     1921 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     1981 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacgc caagaccaca
     2041 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     2101 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     2161 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta gaagcttatc
     2221 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     2281 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     2341 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     2401 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     2461 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     2521 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     2581 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     2641 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     2701 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     2761 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     2821 cgtcgacctc gacccgggcg gccgcttcga gcagacatga taagatacat tgatgagttt
     2881 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2941 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     3001 cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc
     3061 tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca
     3121 tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
     3181 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3241 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcatt aatgaatcgg ccaacgcgcg
     3301 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4141 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4201 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4261 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4321 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4381 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4441 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4501 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4561 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4621 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4681 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4741 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4801 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4861 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4921 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4981 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5041 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5101 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5161 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5221 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     5281 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     5341 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     5401 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     5461 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     5521 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     5581 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     5641 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     5701 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     5761 tgcgggcctc ttcgctatta cgccagctgc gcgctcgctc gctcactgag gccgcccgga
     5821 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
     5881 gagagggagt ggccaactcc atcactaggg gttccttgta gttaatgatt aacccgccat
     5941 gctacttatc tacgtagcca tgctctagga agatctctgc agagggccct gcgtatgcgt
     6001 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     6061 gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     6121 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     6181 caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc
     6241 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
     6301 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
     6361 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggcgcgcgcc aggcggggcg
     6421 gggsggggsg aggggsgggg sggggsgagg cggagaggtg cggcggcagc caatcagagc
     6481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
     6541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
     6601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
     6661 cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga
     6721 tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt
     6781 tttcaggttg gaccggtgcc accatgctgc agaacgagct tgctcttaag ttggctggac
     6841 ttgatattaa caagactgga ggttct
//
