LOCUS       sequence-473746-        6740 bp    DNA     circular SYN 23-MAY-2025
DEFINITION  Cytosol-targeted 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 6740)
  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 6740)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 23, 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-Cyto-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..6740
                     /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"
     CDS             2248..2274
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(2293..2311)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2294..2311)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2331..2348)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2337..2561
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2607..3035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2694..2713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2904..2925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3044..3064)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3049..3378
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3229..3364
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3291..3310
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3445..4239
                     /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(3499..3518)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4109..4128
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4413..4534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4450..4469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4504..4523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4583..4599)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4583..4599)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4596..4618)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4607..4623
                     /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(4631..4637,4638..4655,4656..4661))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4676..4697
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4814..4831)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4985..5573)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5065..5084)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5744..6535,6536..6604))
                     /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     6367..6386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6605..6709)
                     /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 cgaattcctg ccccccctgg agcgcctgac cctgtaatct
     2281 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     2341 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2401 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2461 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2521 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2581 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2701 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2821 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2941 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3001 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3061 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3121 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3181 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3241 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3301 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3361 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3421 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3481 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3541 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3601 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3661 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3721 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3781 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3841 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3901 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3961 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4021 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4081 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4141 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4201 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4261 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4321 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4381 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4441 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4501 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4561 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4621 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4681 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4741 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4801 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4861 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4921 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4981 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5041 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5101 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5161 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5221 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5281 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5341 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5401 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5461 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5521 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5581 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5641 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5701 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5761 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5821 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5881 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5941 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6001 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6061 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6121 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6181 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6241 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6301 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6361 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6421 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6481 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6541 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6601 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6661 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6721 gaaaagtgcc acctgacgtc
//
