LOCUS       sequence-454232-        7392 bp    DNA     circular SYN 15-APR-2025
DEFINITION  FRET-based reporter for monitoring deltaPKC activity at the plasma 
            membrane in cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7392)
  AUTHORS   Kajimoto T, Sawamura S, Tohyama Y, Mori Y, Newton AC
  TITLE     Protein kinase C {delta}-specific activity reporter reveals 
            agonist-evoked nuclear activity controlled by Src family of kinases.
  JOURNAL   J Biol Chem. 2010 Dec 31. 285(53):41896-910.
  PUBMED    20959447
REFERENCE   2  (bases 1 to 7392)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem. 2010 Dec 31. 285(53):41896-910."
COMMENT     Sequence Label: pcDNA3-PM-deltaCKAR
FEATURES             Location/Qualifiers
     source          1..7392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1040..1161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1051..1070)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1105..1124
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1335..2129)
                     /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(1446..1465)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2056..2075
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2196..2525)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2210..2345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2264..2283)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2510..2530
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2539..2967)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2649..2670)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2861..2880
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3226..3243
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        3263..3281
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3263..3280
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(3297..4010,4011..4013))
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(join(4590..5300,5301..5303))
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     HFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     regulatory      5321..5330
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5344..5363)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5345..5363)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5408..5611)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5437..5457)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5612..5991
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6163..6182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6257..6361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6362..6430,6431..7222)
                     /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     complement(6580..6599)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1081 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1141 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1201 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1261 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1321 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1381 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1441 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1501 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1561 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1621 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1681 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1741 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1801 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1861 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1921 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1981 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     2041 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2101 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2161 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2221 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2281 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2341 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2401 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2461 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2521 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2581 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2641 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2701 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2761 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2821 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2881 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2941 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     3001 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3061 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3121 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3181 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3241 aggctgatca gcgagctcta gcatttaggt gacactatag aatagggccc tctagattac
     3301 ttgtacagct cgtccatgcc gtgagtgatc ccggcggcgg tcacgaactc cagcaggacc
     3361 atgtgatcgc gcttctcgtt ggggtctttg ctcagcttgg actggtagct caggtagtgg
     3421 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
     3481 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc
     3541 ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc
     3601 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
     3661 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
     3721 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
     3781 atgtggtcgg ggtagcgggc gaagcacatc aggccgtagc cgaaggtggt cacgagggtg
     3841 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
     3901 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
     3961 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct cacgagaatt
     4021 ccaccagatc caccccgcac ctgtcggagc tgcccaatag cgaaggtgct acggctggac
     4081 acaccccgac ctccggatcc tcctaacttt ttcaccaaat ctttttcttc ggctgtttgc
     4141 ttaagtacta ctctttgttc ttgtaacata cctaatccct cgttaaacag acctgtagta
     4201 tcgttaattt ccactttaaa gccaatgaca aatttattgt ttttatccca aatgatcttg
     4261 atttcatctc cgtcttgtaa aaggaatttc gtaccctgta tcatgcggtt attatttaaa
     4321 tagctcacgt tagttccggt gtggcaatac caaatatcat ctaaaccttg tgccggagat
     4381 tcatacatgc ttttgcctac agcatgcctc tttttgaaaa tgaagcaatg aactcgagac
     4441 aacctactgt cttcaatttt acaattgcaa tcctcggatc taccaatgaa aaatggattc
     4501 acaccttgct gaatctccag gctttcttga ataatgctgt caggcaatgg ttttaaagtt
     4561 aaaaacctac cattaccctt ggtaccagcc ttgtacagct cgtccatgcc gtgagtgatc
     4621 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     4681 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     4741 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     4801 cggatcttga agtgggcctt gatgccgttc ttctgcttgt cggcggtgat atagacgttg
     4861 tggctgatgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     4921 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     4981 gtcttgtagt tgccgtcgtc cttgaagaag atggtacgct cctggacgta gccttcgggc
     5041 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     5101 acgccccagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     5161 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     5221 ctgaacctgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     5281 aacagctcct cgcccttgct caccttcgac ttgatacaac ccatggtggc gaagcttggg
     5341 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     5401 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     5461 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     5521 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     5581 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     5641 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     5701 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     5761 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     5821 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     5881 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     5941 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     6001 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     6061 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     6121 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     6181 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     6241 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6301 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6361 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6421 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6481 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6541 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6601 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6661 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6721 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6781 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6841 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6901 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6961 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7021 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7081 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7141 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7201 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7261 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7321 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7381 caaaggatct tc
//
