LOCUS       sequence-473794-        6752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  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 6752)
  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 6752)
  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-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..6752
                     /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"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     1293..1314
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1420..1441)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1681..1700)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2305..2323)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2306..2323)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2343..2360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2349..2573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2619..3047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2706..2725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2916..2937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3056..3076)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3061..3390
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3241..3376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3303..3322
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3457..4251
                     /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(3511..3530)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4121..4140
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4425..4546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4462..4481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4516..4535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4595..4611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4595..4611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4608..4630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4619..4635
                     /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(4643..4649,4650..4667,4668..4673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4688..4709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4826..4843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4997..5585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5077..5096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5756..6547,6548..6616))
                     /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     6379..6398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6617..6721)
                     /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 tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat gcgattcaga
     1021 agattccaga cgttgaagga caaggctaag gctggcggca ccggcggcag cgagctcagc
     1081 agctacaacg tctatatcaa ggccgacaag cagaagaacg gcatcaaggc gaacttcaag
     1141 atccgccaca acatcgagga cggcggcgtg cagctcgcct accactacca gcagaacacc
     1201 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcgt gcagtccaaa
     1261 ctttcgaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1321 gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccggagg gagcatggtg
     1381 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1441 gtaaacggcc acaagttcag cgtgtccggc gagggtgagg gcgatgccac ctacggcaag
     1501 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1561 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1621 gacttcttca agtccgccat gcccgaaggc tacatccagg agcgcaccat cttcttcaag
     1681 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1741 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1801 gagtacaaca tctcccataa gttttctcaa gaacagatcg gcgaaaacat tgtgtgcagg
     1861 gtcatttgta ccacgggtca aattcccatc cgagatttgt cagctgatat ttcacaagtg
     1921 cttaaggaaa aacgatccat aaagaaagtt tggacatttg gtagaaaccc agcctgtgac
     1981 tatcatttag gaaacatttc aagactgtca aataagcatt tccaaatact actaggagaa
     2041 gacggtaacc ttttattgaa tgacatttcc actaatggga cctggttaaa tgggcaaaaa
     2101 gtcgagaaga acagcaatca gttactgtct caaggtgatg aaataaccgt tggtgtaggc
     2161 gtggaatcag atattttatc tctggtcatt ttcataaacg acaaatttaa gcagtgcctc
     2221 gagcagaaca aagttgatcg ctaagaattc tgcagatatc catcacactg gcggccgctc
     2281 gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     2341 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2401 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2461 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2521 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2581 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2641 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2701 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2761 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2821 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2881 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2941 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3001 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3061 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3121 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3181 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3241 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3301 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3361 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3421 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3481 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3541 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3601 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3661 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3721 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3781 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3841 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3901 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3961 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4021 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4081 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4141 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4201 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4261 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4321 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4381 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4561 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4621 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4681 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4801 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4861 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4921 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4981 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5041 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5101 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5161 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5221 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5281 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5341 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5401 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5461 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5521 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5581 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5641 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5701 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5761 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5821 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5881 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5941 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6001 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6061 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6121 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6181 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6241 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6301 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6361 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6421 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6481 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6541 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6601 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6661 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6721 gcacatttcc ccgaaaagtg ccacctgacg tc
//
