LOCUS       sequence-473790-        6768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enhanced excitation-ratiometric biosensor for monitoring Protein 
            Kinase C activity in living cells, targeted to ER surface..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6768)
  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 6768)
  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-ER-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..6768
                     /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     1309..1330
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1436..1457)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1697..1716)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(2321..2339)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2322..2339)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2359..2376)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2365..2589
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2635..3063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2722..2741)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2932..2953
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3072..3092)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3077..3406
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3257..3392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3319..3338
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3473..4267
                     /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(3527..3546)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4137..4156
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4441..4562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4478..4497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4532..4551
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4611..4627)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4611..4627)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4624..4646)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4635..4651
                     /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(4659..4665,4666..4683,4684..4689))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4704..4725
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4842..4859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5013..5601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5093..5112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5772..6563,6564..6632))
                     /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     6395..6414
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6633..6737)
                     /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 gcttggtacc
      901 ccttgcagct tccagttgtc aggaagagaa ggctcccatg gaccctgtgg tggtgctggg
      961 gctctgtctc tcctgtttgc ttctcctttc actctggaaa cagagctatg ggggagggga
     1021 tcccatgcga ttcagaagat tccagacgtt gaaggacaag gctaaggctg gcggcaccgg
     1081 cggcagcgag ctcagcagct acaacgtcta tatcaaggcc gacaagcaga agaacggcat
     1141 caaggcgaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgcctacca
     1201 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1261 gagcgtgcag tccaaacttt cgaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1321 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agggcggtac
     1381 cggagggagc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1441 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gtgagggcga
     1501 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1561 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1621 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctaca tccaggagcg
     1681 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1741 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1801 cctggggcac aagctggagt acaacatctc ccataagttt tctcaagaac agatcggcga
     1861 aaacattgtg tgcagggtca tttgtaccac gggtcaaatt cccatccgag atttgtcagc
     1921 tgatatttca caagtgctta aggaaaaacg atccataaag aaagtttgga catttggtag
     1981 aaacccagcc tgtgactatc atttaggaaa catttcaaga ctgtcaaata agcatttcca
     2041 aatactacta ggagaagacg gtaacctttt attgaatgac atttccacta atgggacctg
     2101 gttaaatggg caaaaagtcg agaagaacag caatcagtta ctgtctcaag gtgatgaaat
     2161 aaccgttggt gtaggcgtgg aatcagatat tttatctctg gtcattttca taaacgacaa
     2221 atttaagcag tgcctcgagc agaacaaagt tgatcgctaa gaattctgca gatatccatc
     2281 acactggcgg ccgctcgagc atgcatctag agggccctat tctatagtgt cacctaaatg
     2341 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2401 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2461 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2521 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2581 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2641 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2701 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2761 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2821 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2881 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2941 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3001 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3061 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3181 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3241 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3301 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3361 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3421 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3481 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3541 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     3601 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     3661 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     3721 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     3781 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     3841 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3901 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3961 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4021 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4081 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4141 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4201 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4261 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4321 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4381 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4441 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4501 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4561 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     4621 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4681 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4741 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4801 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4861 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4921 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4981 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5041 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5101 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5161 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5221 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5281 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5341 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5401 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5461 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5521 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5581 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5641 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5701 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5761 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5821 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5881 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5941 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6001 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6061 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6121 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6181 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6241 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6301 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6361 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6421 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6481 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6541 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6601 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6661 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6721 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
