LOCUS       sequence-313438-        7511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Improved FRET-based biosensor for monitoring Protein Kinase C 
            activity..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7511)
  AUTHORS   Ross BL, Tenner B, Markwardt ML, Zviman A, Shi G, Kerr JP, Snell NE,
            McFarland JJ, Mauban JR, Ward CW, Rizzo MA, Zhang J
  TITLE     Single-color, ratiometric biosensors for detecting signaling 
            activities in live cells.
  JOURNAL   eLife. 2018 Jul 3;7. pii: 35458. doi: 10.7554/eLife.35458
  PUBMED    29968564
REFERENCE   2  (bases 1 to 7511)
  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: "eLife."; 
            date: "2018-07-3"; volume: "7. pii: 35458. doi"; pages: " 
            10.7554/eLife.3545"
COMMENT     Sequence Label: pcDNA3-CKAR2
FEATURES             Location/Qualifiers
     source          1..7511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        132..511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        512..715
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     666..686
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     760..779
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        760..778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      798..807
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             819..836
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             840..872
                     /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(876..884,885..899)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(906..908,909..911,912..1622)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2163..2897
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     promoter        complement(2961..2979)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2962..2979)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2999..3016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3005..3229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3275..3703
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3362..3381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3572..3593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3712..3732)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3717..4046
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3897..4032
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3959..3978
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4113..4907
                     /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(4167..4186)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4777..4796
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5081..5202
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5118..5137)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5172..5191
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5251..5267)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5251..5267)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5264..5286)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5275..5291
                     /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(5299..5305,5306..5323,5324..5329))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5344..5365
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5482..5499)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5653..6241)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5733..5752)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6412..7203,7204..7272))
                     /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     7035..7054
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7273..7377)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7452..7471)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
       61 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      121 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      181 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      241 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      301 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      361 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      421 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      481 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      541 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      601 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      661 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
      721 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
      781 gacccaagct tgcggccgcc accatggtgc ggggttctca tcatcatcat catcatggta
      841 tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat gacgataagg
      901 atcccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      961 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1021 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1081 ccaccctcgt gaccaccctg agctggggcg tgcagtgctt cgcccgctac cccgaccaca
     1141 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1201 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1261 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1321 ggcacaagct ggagtacaac gccatccacg gcaacgtcta tatcaccgcc gacaagcaga
     1381 agaacggcat caaggccaac ttcggcctca actgcaacat cgaggacggc agcgtgcagc
     1441 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1501 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1561 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1621 agcgcatgca taagttttct caagaacaga tcggcgaaaa cattgtgtgc agggtcattt
     1681 gtaccacggg tcaaattccc atccgagatt tgtcagctga tatttcacaa gtgcttaagg
     1741 aaaaacgatc cataaagaaa gtttggacat ttggtagaaa cccagcctgt gactatcatt
     1801 taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga gaagacggta
     1861 accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa aaagtcgaga
     1921 agaacagcaa tcagttactg tctcaaggtg atgaaataac cgttggtgta ggcgtggaat
     1981 cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc ctcgagcaga
     2041 acaaagttga tcgctctgca ggtaagccag gcagcggcga gggcagcacc aagggccgat
     2101 tcagaagatt ccagacgttg aaggacaagg ctaaggccgg cggcaccggc ggcagcgagc
     2161 tcatggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2221 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     2281 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2341 tcggcatgga cgagctgtac aagggtggca gcggtggcat ggtgagcaag ggcgaggagc
     2401 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2461 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga
     2521 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg
     2581 gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2641 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2701 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2761 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2821 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     2881 tccgccacaa catcgagtaa gaattctgca gatatccatc acactggcgg ccgctcgagc
     2941 atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc
     3001 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3061 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3121 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3181 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3241 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3301 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3361 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3421 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3481 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3541 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3601 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3661 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     3721 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3781 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3841 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3901 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3961 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4021 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4081 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4141 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4201 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4261 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4321 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4381 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4441 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4501 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4561 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4621 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4681 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4741 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4801 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4861 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4921 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4981 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5041 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5101 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5161 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5221 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5281 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5341 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5401 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5461 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5521 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5581 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5641 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5701 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5761 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5821 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5881 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5941 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6001 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6061 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6121 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6181 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6241 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6301 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6361 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6421 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6481 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6541 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6601 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6661 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6721 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6781 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6841 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6901 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6961 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7021 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7081 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7141 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7201 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7261 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7321 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7381 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7441 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7501 tgtgtgttgg a
//
