LOCUS       sequence-473738-        6689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted enhanced excitation-ratiometric biosensor 
            for monitoring Protein Kinase C activity in living cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6689)
  AUTHORS   Su Q, Zhang J, Lin W, Zhang JF, Newton AC, Mehta S, Yang J, Zhang J
  TITLE     Sensitive fluorescent biosensor reveals differential subcellular 
            regulation of PKC.
  JOURNAL   Nat Chem Biol. 2024 Oct 11. doi: 10.1038/s41589-024-01758-3.
  PUBMED    39394268
REFERENCE   2  (bases 1 to 6689)
  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. 2024 Oct 11. doi: 10.1038/s41589-024-01758-3."
COMMENT     Sequence Label: pcDNA3-PM-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..6689
                     /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"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1230..1251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1357..1378)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1618..1637)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2242..2260)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2243..2260)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2280..2297)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2286..2510
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2556..2984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2643..2662)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2853..2874
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2993..3013)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2998..3327
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3178..3313
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3240..3259
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3394..4188
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3448..3467)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4058..4077
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4362..4483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4399..4418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4453..4472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4532..4548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4532..4548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4545..4567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4556..4572
                     /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(4580..4586,4587..4604,4605..4610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4625..4646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4763..4780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4934..5522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5014..5033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5693..6484,6485..6553))
                     /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     6316..6335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6554..6658)
                     /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 gcttgcggcc
      901 gccaccatgg gctgcatcaa gagcaagcgc aaggacaagg atcccatgcg attcagaaga
      961 ttccagacgt tgaaggacaa ggctaaggct ggcggcaccg gcggcagcga gctcagcagc
     1021 tacaacgtct atatcaaggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc
     1081 cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc actaccagca gaacaccccc
     1141 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcgtgca gtccaaactt
     1201 tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1261 gggatcactc tcggcatgga cgagctgtac aagggcggta ccggagggag catggtgagc
     1321 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1381 aacggccaca agttcagcgt gtccggcgag ggtgagggcg atgccaccta cggcaagctg
     1441 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1501 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1561 ttcttcaagt ccgccatgcc cgaaggctac atccaggagc gcaccatctt cttcaaggac
     1621 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1681 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1741 tacaacatct cccataagtt ttctcaagaa cagatcggcg aaaacattgt gtgcagggtc
     1801 atttgtacca cgggtcaaat tcccatccga gatttgtcag ctgatatttc acaagtgctt
     1861 aaggaaaaac gatccataaa gaaagtttgg acatttggta gaaacccagc ctgtgactat
     1921 catttaggaa acatttcaag actgtcaaat aagcatttcc aaatactact aggagaagac
     1981 ggtaaccttt tattgaatga catttccact aatgggacct ggttaaatgg gcaaaaagtc
     2041 gagaagaaca gcaatcagtt actgtctcaa ggtgatgaaa taaccgttgg tgtaggcgtg
     2101 gaatcagata ttttatctct ggtcattttc ataaacgaca aatttaagca gtgcctcgag
     2161 cagaacaaag ttgatcgcta agaattctgc agatatccat cacactggcg gccgctcgag
     2221 catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc
     2281 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2341 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2401 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2461 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2521 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2581 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2641 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2701 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2761 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2821 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2881 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2941 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3001 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3061 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3121 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3181 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3241 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3301 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3361 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3421 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3481 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3541 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg
     3601 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3661 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3721 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3781 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3841 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3901 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc
     3961 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4021 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4081 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4141 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4201 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4261 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4321 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4381 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4441 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4501 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4561 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4621 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4681 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4741 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4801 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4861 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4921 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4981 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5041 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5101 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5161 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5221 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5281 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5341 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5401 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5461 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5521 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5581 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5641 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5701 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5761 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5821 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5881 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5941 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6001 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6061 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6121 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6181 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6241 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6301 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6361 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6421 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6481 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6541 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6661 catttccccg aaaagtgcca cctgacgtc
//
