LOCUS       sequence-473748-        7925 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enhanced excitation-ratiometric biosensor for monitoring Protein 
            Kinase C activity in living cells, targeted to lysosome surface..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7925)
  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 7925)
  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-Lyso-ExRai-CKAR2
FEATURES             Location/Qualifiers
     source          1..7925
                     /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"
     regulatory      905..914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(907..990,991..2157)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFAYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTPHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     primer_bind     2466..2487
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2593..2614)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2854..2873)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3478..3496)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3479..3496)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3516..3533)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3522..3746
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3792..4220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3879..3898)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4089..4110
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4229..4249)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4234..4563
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4414..4549
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4476..4495
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4630..5424
                     /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(4684..4703)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5294..5313
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5598..5719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5635..5654)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5689..5708
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5768..5784)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5768..5784)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5781..5803)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5792..5808
                     /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(5816..5822,5823..5840,5841..5846))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5861..5882
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5999..6016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6170..6758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6250..6269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6929..7720,7721..7789))
                     /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     7552..7571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7790..7894)
                     /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 cggcgcccgg cagcgcccgg cgacccctgc tgctgctact gctgttgctg
      961 ctgctcggcc tcatgcattg tgcgtcagca gcaatgttta tggtgaaaaa tggcaacggg
     1021 accgcgtgca taatggccaa cttctctgct gccttctcag tgaactacga caccaagagt
     1081 ggccctaaga acatgacctt tgacctgcca tcagatgcca cagtggtgct caaccgcagc
     1141 tcctgtggaa aagagaacac ttctgacccc agtctcgtga ttgcttttgg aagaggacat
     1201 acactcactc tcaatttcac gagaaatgca acacgttaca gcgtccagct catgagtttt
     1261 gcttataact tgtcagacac acaccttttc cccaatgcga gctccaaaga aatcaagact
     1321 gtggaatcta taactgacat cagggcagat atagataaaa aatacagatg tgttagtggc
     1381 acccaggtcc acatgaacaa cgtgaccgta acgccccatg atgccaccat ccaggcgtac
     1441 ctttccaaca gcagcttcag caggggagag acacgctgtg aacaagacag gccttcccca
     1501 accacagcgc cccctgcgcc acccagcccc tcgccctcac ccgtgcccaa gagcccctct
     1561 gtggacaagt acaacgtgag cggcaccaac gggacctgcc tgctggccag catggggctg
     1621 cagctgaacc tcacctatga gaggaaggac aacacgacgg tgacaaggct tctcaacatc
     1681 aaccccaaca agacctcggc cagcgggagc tgcggcgccc acctggtgac tctggagctg
     1741 cacagcgagg gcaccaccgt cctgctcttc cagttcggga tgaatgcaag ttctagccgg
     1801 tttttcctac aaggaatcca gttgaataca attcttcctg acgccagaga ccctgccttt
     1861 aaagctgcca acggctccct gcgagcgctg caggccacag tcggcaattc ctacaagtgc
     1921 aacgcggagg agcacgtccg tgtcacgaag gcgttttcag tcaatatatt caaagtgtgg
     1981 gtccaggctt tcaaggtgga aggtggccag tttggctctg tggaggagtg tctgctggac
     2041 gagaacagca tgctgatccc catcgctgtg ggtggtgccc tggcggggct ggtcctcatc
     2101 gtcctcatcg cctacctcgt cggcaggaag aggagtcacg caggctacca gactatcggt
     2161 ggtggcggtg gtgcggatcc catgcgattc agaagattcc agacgttgaa ggacaaggct
     2221 aaggctggcg gcaccggcgg cagcgagctc agcagctaca acgtctatat caaggccgac
     2281 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcggc
     2341 gtgcagctcg cctaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2401 cccgacaacc actacctgag cgtgcagtcc aaactttcga aagaccccaa cgagaagcgc
     2461 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2521 ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg gcgaggagct gttcaccggg
     2581 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2641 ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2701 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2761 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2821 ggctacatcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2881 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2941 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acatctccca taagttttct
     3001 caagaacaga tcggcgaaaa cattgtgtgc agggtcattt gtaccacggg tcaaattccc
     3061 atccgagatt tgtcagctga tatttcacaa gtgcttaagg aaaaacgatc cataaagaaa
     3121 gtttggacat ttggtagaaa cccagcctgt gactatcatt taggaaacat ttcaagactg
     3181 tcaaataagc atttccaaat actactagga gaagacggta accttttatt gaatgacatt
     3241 tccactaatg ggacctggtt aaatgggcaa aaagtcgaga agaacagcaa tcagttactg
     3301 tctcaaggtg atgaaataac cgttggtgta ggcgtggaat cagatatttt atctctggtc
     3361 attttcataa acgacaaatt taagcagtgc ctcgagcaga acaaagttga tcgctaagaa
     3421 ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg gccctattct
     3481 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     3541 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3601 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3661 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3721 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3781 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3841 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3901 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3961 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4021 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4081 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4141 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4201 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4261 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4321 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4381 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4441 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4501 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4561 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     4621 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4681 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4741 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4801 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4861 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4921 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4981 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     5041 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     5101 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     5161 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     5221 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     5281 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     5341 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5401 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5461 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5521 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5581 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5641 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5701 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5761 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5821 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5881 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5941 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6001 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6061 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6121 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6181 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6241 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6301 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6361 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6421 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6481 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6541 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6601 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6661 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6721 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6781 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6841 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6901 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6961 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7021 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7081 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7141 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7201 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7261 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7321 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7381 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7441 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7501 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7561 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7621 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7681 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7741 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7801 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7861 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7921 acgtc
//
