LOCUS       sequence-224339-        3363 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Mammalian expression of the AKAR5' sensor C-terminally fused to the 
            C-terminus of KRas (membrane targeting) for detecting PKA activity 
            using two-photon fluorescence lifetime imaging microscopy (2pFLIM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3363)
  AUTHORS   Tillo SE, Xiong WH, Takahashi M, Miao S, Andrade AL, Fortin DA, Yang
            G, Qin M, Smoody BF, Stork PJS, Zhong H
  TITLE     Liberated PKA Catalytic Subunits Associate with the Membrane via 
            Myristoylation to Preferentially Phosphorylate Membrane Substrates.
  JOURNAL   Cell Rep. 2017 Apr 18;19(3):617-629. doi: 
            10.1016/j.celrep.2017.03.070.
  PUBMED    28423323
REFERENCE   2  (bases 1 to 3363)
  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: 
            "10.1016/j.celrep.2017.03"; journalName: "Cell Rep"; date: 
            "2017-04-18- 18"; volume: "19"; issue: "3"; pages: "617-629"
COMMENT     Sequence Label: pCAGGS_m-AKAR5'
FEATURES             Location/Qualifiers
     source          1..3363
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     19..41
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     103..122
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      175..184
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             196..213
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             217..249
                     /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(253..261,262..276)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(283..285,286..288,289..999)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1738..1740,1741..1743,1744..2256)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNWNSVNVYIMADKQKNGIK
                     VNFKIRHNIE"
     intron          2426..2558
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2541..2560
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2567..3155
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2620..2640)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3038..3049)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3182..3199)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3188..3299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
       61 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
      121 tgctgtctca tcattttggc aaagaattcg gtaccgcggg cccagcttgc ggccgccacc
      181 atggtgcggg gttctcatca tcatcatcat catggtatgg ctagcatgac tggtggacag
      241 caaatgggtc gggatctgta cgacgatgac gataaggatc ccatggtgag caagggcgag
      301 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      361 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      421 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
      481 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
      541 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      601 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      661 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
      721 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
      781 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
      841 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
      901 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
      961 gccgccggga tcactctcgg catggacgag ctgtacaagc gcatgcataa gttttctcaa
     1021 gaacagatcg gcgaaaacat tgtgtgcagg gtcatttgta ccacgggtca aattcccatc
     1081 cgagatttgt cagctgatat ttcacaagtg cttaaggaaa aacgatccat aaagaaagtt
     1141 tggacatttg gtagaaaccc agcctgtgac tatcatttag gaaacatttc aagactgtca
     1201 aataagcatt tccaaatact actaggagaa gacggtaacc ttttattgaa tgacatttcc
     1261 actaatggga cctggttaaa tgggcaaaaa gtcgagaaga acagcaatca gttactgtct
     1321 caaggtgatg aaataaccgt tggtgtaggc gtggaatcag atattttatc tctggtcatt
     1381 ttcataaacg acaaatttaa gcagtgcctc gagcagaaca aagttgatcg ctctgcaggt
     1441 aagccaggca gcggcgaggg cagcaccaag ggcctgcgtc gcgccaccct ggtggacggc
     1501 ggcaccggcg gcagcgagct catgggcagc gtgcagctcg ccgaccacta ccagcagaac
     1561 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     1621 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gagagtgacc
     1681 gccgccggga tcactctcgg catggacgag ctatacaagg gtggaactgg aggttcaatg
     1741 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1801 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1861 aagctgaccc tgaagctcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1921 gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct accccgacca catgaagcag
     1981 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2041 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2101 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2161 ctggagtaca actggaacag cgtcaacgtc tatatcatgg ccgacaagca gaagaacggc
     2221 atcaaggtga acttcaagat ccgccacaac atcgagggtg aattcggtaa aaagaagaaa
     2281 aagaagtcaa agacaaagtg tgtaattatg taatctaggg gatccaccgg aagccacctg
     2341 catgcggccg catgatcagc tggatgcatc gatcacgcgt accggtgctc gaggtaccga
     2401 tgaatagcta aggtcgaggc cgcaggtaag tatcaaggtt acaagacagg tttaaggaga
     2461 ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat
     2521 tggtcttact gacatccact ttgcctttct ctccacaggt gtcgacaatc aacctctgga
     2581 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2641 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2701 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     2761 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     2821 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     2881 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     2941 ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac
     3001 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3061 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3121 gacgagtcgg atctcccttt gggccgcctc cccgcctgga attcgagctc ggtacgatca
     3181 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3241 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3301 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3361 gag
//
