LOCUS       sequence-386320-        6744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for GFP tagged PKCA. Kinase-inactive 
            mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6744)
  AUTHORS   Cameron AJ, Escribano C, Saurin AT, Kostelecky B, Parker PJ
  TITLE     PKC maturation is promoted by nucleotide pocket occupation 
            independently of intrinsic kinase activity.
  JOURNAL   Nat Struct Mol Biol. 2009 Jun;16(6):624-30. doi: 10.1038/nsmb.1606. 
            Epub 2009 May 24.
  PUBMED    19465915
REFERENCE   2  (bases 1 to 6744)
  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: "10.1038/nsmb.1606";
            journalName: "Nat Struct Mol Biol"; date: "2009-06"; volume: "16"; 
            issue: "6"; pages: "624-30"
COMMENT     Sequence Label: GFP-PKCA D463N
FEATURES             Location/Qualifiers
     source          1..6744
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             159..953
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             159..953
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(213..232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     823..842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1141..1160)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1185..1232
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1561..2149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2050..2069
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2324..2627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2628..2831
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2782..2802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2870..2879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2876..2878,2879..2881,2882..3592)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2876..2878,2879..2881,2882..3592)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2921..2942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3182..3201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3529..3550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5795..5916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5832..5851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5886..5905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5923..6378)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6060..6081)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6272..6291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6405..6509
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6511..6744,1..124)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6511..6702
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6534..6554)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6719..6744,1..110)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
       61 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
      121 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
      181 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      241 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      301 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
      361 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      421 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      481 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      541 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
      601 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
      661 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
      721 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
      781 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
      841 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
      901 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
      961 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1021 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1081 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     1141 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     1201 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     1261 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     1321 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     1381 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     1441 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     1501 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1561 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1621 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1681 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1741 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1801 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1861 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1921 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1981 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2041 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2101 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2161 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2221 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     2281 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     2341 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     2401 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2461 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2521 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     2581 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     2641 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     2701 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2761 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2821 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
     2881 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     2941 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     3001 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     3061 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     3121 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     3181 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     3241 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     3301 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     3361 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     3421 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3481 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     3541 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggccg
     3601 gactcagatc tcgagctcaa gcttcgaatt catggctgac gtcttcccgg ccgccgagcc
     3661 ggcggcgccg caggacgtgg ccaaccgctt cgcccgcaaa ggggcgctga ggcagaagaa
     3721 cgtgcacgag gtgaagaacc accgcttcat cgcgcgcttc ttcaagcagc ccaccttctg
     3781 cagccactgc accgacttca tctgggggtt tgggaaacaa ggcttccagt gccaagtttg
     3841 ctgttttgtg gttcacaaga ggtgccatga atttgttact ttttcttgtc cgggggcgga
     3901 taaaggaccc gacacagatg acccgaggag caagcacaag ttcaagatcc acacgtatgg
     3961 cagccccacc ttctgtgatc actgcggctc cctgctgtac ggactcatcc accaggggat
     4021 gaaatgtgac acctgtgata tgaacgtgca caagcagtgc gtgatcaacg tgcccagcct
     4081 ctgcgggatg gaccacacgg agaagagggg ccgcatctac ctgaaggccg aggtcacgga
     4141 tgaaaagctg cacgtcacag tacgagacgc gaaaaaccta atccctatgg atccaaatgg
     4201 gctttcagat ccttacgtga agctgaagct tattcctgac cccaagaacg agagcaaaca
     4261 gaaaaccaag accatccgct cgacgctgaa cccccggtgg gacgagtcct tcacgttcaa
     4321 attaaaacct tctgataaag accggcgact gtccgtggaa atctgggact gggatcgaac
     4381 cacacggaac gacttcatgg ggtccctttc ctttggggtc tcggagctga tgaagatgcc
     4441 ggccagcgga tggtacaagc tgctgaacca agaggagggc gagtactaca acgtgccgat
     4501 ccccgaaggc gacgaggaag gcaatgtgga gctcaggcag aaattcgaga aagccaagct
     4561 tggccctgcc ggcaacaaag tcatcagtcc ctccgaggac aggagacagc cttccaacaa
     4621 cctgggcaga gtgaagctca cggacttcaa cttcctcatg gtgctgggca aaggcagctt
     4681 tgggaaggtg atgctggccg accgaaaggg gacagaggag ctgtacgcca tcaagatcct
     4741 gaagaaggac gtggtcatcc aggacgacga cgtggagtgc accatggtgg agaagcgggt
     4801 cctggcgctg ctcgacaagc cgccgttcct gacgcagctg cactcctgct tccagacggt
     4861 ggaccggctg tacttcgtca tggagtacgt caacggcggg gacctcatgt accacatcca
     4921 gcaggtcggg aagttcaagg agccgcaagc agtgttctat gcagcagaga tttccatcgg
     4981 gctgttcttt cttcataaaa gaggaatcat ttatcggaac ctgaagttag acaacgtcat
     5041 gctggactcg gaaggacaca ttaagatcgc ggacttcggg atgtgcaagg agcacatgat
     5101 ggacggcgtc acgaccagga ccttctgcgg gacccccgac tacatcgccc cagagataat
     5161 cgcctatcag ccgtacggga agtccgtgga ctggtgggcc tacggcgtcc tgttgtacga
     5221 gatgttggcc gggcagcctc cgttcgacgg cgaggacgag gacgagctgt tccagtccat
     5281 catggagcac aacgtctcgt accccaagtc cttgtccaag gaggccgtgt ccatctgcaa
     5341 agggctgatg accaagcacc ccgggaagcg gctgggctgc gggcccgagg gcgagcgcga
     5401 cgtgcgggag catgccttct tccggaggat cgactgggag aagctggaga accgtgagat
     5461 ccagccaccc ttcaagccca aagtgtgcgg caaaggagca gagaactttg acaagttctt
     5521 cacgcgaggg cagcctgtct tgacgccgcc cgaccagctg gtcatcgcta acatcgacca
     5581 gtctgatttt gaaggcttct cctacgtcaa cccccagttc gtgcacccca tcctgcagag
     5641 cgcggtatga ggtaccgcgg gcccgggatc caccggatct agataactga tcataatcag
     5701 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     5761 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     5821 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5881 tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta agcgttaata
     5941 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     6001 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     6061 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     6121 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     6181 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     6241 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     6301 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     6361 cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6421 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6481 tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt
     6541 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     6601 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     6661 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     6721 cccgccccta actccgccca gttc
//
