LOCUS       sequence-473796-        7832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Subcellularly targeted enhanced excitation-ratiometric biosensor for
            monitoring Protein Kinase C activity in living cells for selective 
            detection of aPKC activity within the Par complex..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7832)
  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 7832)
  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-Par6-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..7832
                     /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"
     primer_bind     2401..2422
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     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     complement(3423..3440)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3429..3653
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3699..4127
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3786..3805)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3996..4017
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4136..4156)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4141..4470
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4321..4456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4383..4402
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4537..5331
                     /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(4591..4610)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5201..5220
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5505..5626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5542..5561)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5596..5615
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5675..5691)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5675..5691)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5688..5710)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5699..5715
                     /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(5723..5729,5730..5747,5748..5753))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5768..5789
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5906..5923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6077..6665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6157..6176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6836..7627,7628..7696))
                     /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     7459..7478
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7697..7801)
                     /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 aagcttatgg cccggccgca gaggactccg gcgcgcagtc ccgatagcat
      961 cgtcgaggtg aagagcaaat ttgacgccga gttccgacgc ttcgcgctgc ctcgcgcttc
     1021 ggtgagcggc ttccaggagt tctcgcggtt gctgcgggcg gtgcaccaga tcccgggcct
     1081 ggacgtgcta cttggctata cggatgctca tggcgacctg ctgcccctca ccaacgacga
     1141 cagcctgcac cgggccctgg ccagcgggcc cccgccactg cgcctactgg tgcagaagcg
     1201 ggcagaagct gactccagcg gcctggcttt tgcctccaac tctctgcagc ggcgcaagaa
     1261 agggctcttg ctgcggccag tggcacccct gcgcacccgg ccacccttgc taatcagcct
     1321 gccccaagat ttccgccagg tttcctcagt catagacgtg gacctactgc ctgagaccca
     1381 ccgacgggtg cggctgcaca agcatggttc agaccgcccc ctgggcttct acatccgaga
     1441 tggcatgagc gtgcgtgtgg ctccccaggg cctggagcgg gttccaggaa tcttcatctc
     1501 ccgcctggta cgtgggggtc tggctgagag tacagggctg ctggcggtca gtgatgagat
     1561 cctcgaggtc aatggcattg aagtagccgg gaagaccttg gaccaagtga cggacatgat
     1621 ggttgccaac agccataacc tcattgtcac tgtcaagccc gccaaccagc gcaataacgt
     1681 ggtgcgaggg gcatctgggc gtttgacagg tcctccctct gcagggcctg ggcctgctga
     1741 gcctgatagt gacgatgaca gcagtgacct ggtcattgag aaccgccagc ctcccagttc
     1801 caatgggctg tctcaggggc ccccgtgctg ggacctgcac cctggctgcc gacatcctgg
     1861 tacccgcagc tctctgccct ccctggatga ccaggagcag gccagttctg gctgggggag
     1921 tcgcattcga ggagatggta gtggcttcag cctctccgga ggtaccagtg ctggtggtag
     1981 tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggttccg gcagtgctgg
     2041 tggtagtgct ggtggtagta ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag
     2101 tgctggtggt ggatccatgc gattcagaag attccagacg ttgaaggaca aggctaaggc
     2161 tggcggcacc ggcggcagcg agctcagcag ctacaacgtc tatatcaagg ccgacaagca
     2221 gaagaacggc atcaaggcga acttcaagat ccgccacaac atcgaggacg gcggcgtgca
     2281 gctcgcctac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     2341 caaccactac ctgagcgtgc agtccaaact ttcgaaagac cccaacgaga agcgcgatca
     2401 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     2461 caagggcggt accggaggga gcatggtgag caagggcgag gagctgttca ccggggtggt
     2521 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2581 gggtgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2641 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     2701 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2761 catccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     2821 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2881 ggacggcaac atcctggggc acaagctgga gtacaacatc tcccataagt tttctcaaga
     2941 acagatcggc gaaaacattg tgtgcagggt catttgtacc acgggtcaaa ttcccatccg
     3001 agatttgtca gctgatattt cacaagtgct taaggaaaaa cgatccataa agaaagtttg
     3061 gacatttggt agaaacccag cctgtgacta tcatttagga aacatttcaa gactgtcaaa
     3121 taagcatttc caaatactac taggagaaga cggtaacctt ttattgaatg acatttccac
     3181 taatgggacc tggttaaatg ggcaaaaagt cgagaagaac agcaatcagt tactgtctca
     3241 aggtgatgaa ataaccgttg gtgtaggcgt ggaatcagat attttatctc tggtcatttt
     3301 cataaacgac aaatttaagc agtgcctcga gcagaacaaa gttgatcgct aagaattctg
     3361 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     3421 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3481 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3541 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3601 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3661 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3721 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3781 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3841 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3901 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3961 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4021 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4081 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4141 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4201 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4261 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4321 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4381 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4441 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4501 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4561 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4621 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4681 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4741 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4801 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4861 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4921 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4981 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     5041 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     5101 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5161 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5221 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5281 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5341 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5401 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5461 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5521 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5581 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5641 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5701 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5761 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5821 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5881 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5941 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     6001 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6061 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6121 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6181 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6241 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6301 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6361 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6421 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6481 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6541 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6601 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6661 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6721 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6781 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6841 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6901 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6961 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     7021 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7081 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7141 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7201 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7261 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7321 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7381 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7441 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7501 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7561 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7621 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7681 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7741 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7801 gcacatttcc ccgaaaagtg ccacctgacg tc
//
