LOCUS       sequence-307411-        6287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes negative-control C-terminal (substrate) fragment of 
            FRET-based bimolecular PKC activity reporter (bimCKAR); cytosol 
            targeted. Use in conjunction with pcDNA3-Cerulean-FHA1-NES.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6287)
  AUTHORS   Herbst KJ, Allen MD, Zhang J
  TITLE     Luminescent kinase activity biosensors based on a versatile 
            bimolecular switch.
  JOURNAL   J Am Chem Soc. 2011 Apr 20;133(15):5676-9. doi: 10.1021/ja1117396. 
            Epub 2011 Mar 25.
  PUBMED    21438554
REFERENCE   2  (bases 1 to 6287)
  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"; doi: "10.1021/ja1117396";
            journalName: "J Am Chem Soc"; date: "2011-04-20- 20"; volume: "133";
            issue: "15"; pages: "5676-9"
COMMENT     Sequence Label: pcDNA3-PKCsub(T/A)-YPet-NES
FEATURES             Location/Qualifiers
     source          1..6287
                     /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     complement(1378..1397)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1795..1821
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(1840..1858)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1841..1858)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1878..1895)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1884..2108
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2154..2582
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2241..2260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2451..2472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2591..2611)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2596..2925
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2776..2911
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2838..2857
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2992..3786
                     /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(3046..3065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3656..3675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3960..4081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3997..4016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4051..4070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4130..4146)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4130..4146)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4143..4165)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4154..4170
                     /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(4178..4184,4185..4202,4203..4208))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4223..4244
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4361..4378)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4532..5120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4612..4631)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5291..6082,6083..6151))
                     /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     5914..5933
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6152..6256)
                     /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 agattccagg ccttgaagga caaggctaag gccggcggca gcggcggcag catggtgagc
     1081 aagggcgagg agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
     1141 aacggccaca agttcagcgt gagcggcgag ggcgagggcg acgccaccta cggcaagctg
     1201 accctgaagc tgctgtgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
     1261 accctgggct acggcgtgca gtgcttcgcc cggtaccccg accacatgaa gcagcacgac
     1321 ttcttcaaga gcgccatgcc cgagggctac gtgcaggagc ggaccatctt cttcaaggac
     1381 gacggcaact acaagacccg ggccgaggtg aagttcgagg gcgacaccct ggtgaaccgg
     1441 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctgggcca caagctggag
     1501 tacaactaca acagccacaa cgtgtacatc accgccgaca agcagaagaa cggcatcaag
     1561 gccaacttca agatccggca caacatcgag gacggcggcg tgcagctggc cgaccactac
     1621 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1681 taccagagcg ccctgttcaa ggaccccaac gagaagcggg accacatggt gctgctggag
     1741 ttcctgaccg ccgccggcat caccgagggc atggacgagc tgtacaagga attcctgccc
     1801 cccctggagc gcctgaccct gtaatctaga gggccctatt ctatagtgtc acctaaatgc
     1861 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1921 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1981 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2041 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2101 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2161 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2221 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2281 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2341 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2401 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2461 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2521 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2581 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2641 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2701 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2761 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2821 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2881 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     2941 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3001 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3061 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3121 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3181 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3241 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3301 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3361 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3421 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3481 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3541 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3601 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3661 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3721 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3781 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3841 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3901 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     3961 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4021 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4081 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4141 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4201 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4261 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4321 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4381 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4441 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4501 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4561 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4621 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4681 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4741 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4801 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4861 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4921 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4981 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5041 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5101 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5161 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5221 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5281 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5341 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5401 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5461 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5521 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5581 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5641 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5701 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5761 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5821 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5881 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5941 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6001 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6061 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6121 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6181 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6241 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
