LOCUS       sequence-215469-        8082 bp    DNA     circular SYN 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      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8082)
  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 8082)
  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..8082
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1750..1759
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1771..1788
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1792..1824
                     /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(1828..1836,1837..1851)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1858..1860,1861..1863,1864..2574)
                     /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(3313..3315,3316..3318,3319..3831)
                     /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          4005..4138
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4121..4140
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4147..4735
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4200..4220)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4618..4629)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4762..4779)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4768..4879
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5106..5125)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5171..5226
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5225..5244)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5585..5601)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5598..5620)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5609..5625
                     /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(5633..5639,5640..5657,5658..5663))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5678..5699
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5758..5953
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5804..5939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5959..6093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5996..6015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6050..6069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6161..6178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6332..6920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6412..6431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7091..7882,7883..7951))
                     /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     7714..7733
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7952..8056)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg ggcgagggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcggtacc gcgggcccag
     1741 cttgcggccg ccaccatggt gcggggttct catcatcatc atcatcatgg tatggctagc
     1801 atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatcccatg
     1861 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1921 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1981 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2041 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2101 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2161 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2221 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2281 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2341 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2401 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2461 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2521 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcgcatg
     2581 cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg
     2641 ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga
     2701 tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac
     2761 atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta
     2821 ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc
     2881 aatcagttac tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt
     2941 ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt
     3001 gatcgctctg caggtaagcc aggcagcggc gagggcagca ccaagggcct gcgtcgcgcc
     3061 accctggtgg acggcggcac cggcggcagc gagctcatgg gcagcgtgca gctcgccgac
     3121 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3181 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3241 ctggagagag tgaccgccgc cgggatcact ctcggcatgg acgagctata caagggtgga
     3301 actggaggtt caatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     3361 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     3421 gatgccacct acggcaagct gaccctgaag ctcatctgca ccaccggcaa gctgcccgtg
     3481 ccctggccca ccctcgtgac caccttcggc tacggcctga tgtgcttcgc ccgctacccc
     3541 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3601 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3661 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3721 atcctggggc acaagctgga gtacaactgg aacagcgtca acgtctatat catggccgac
     3781 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga gggtgaattc
     3841 ggtaaaaaga agaaaaagaa gtcaaagaca aagtgtgtaa ttatgtaatc taggggatcc
     3901 accggaagcc acctgcatgc ggccgcatga tcagctggat gcatcgatca cgcgtaccgg
     3961 tgctcgaggt accgatgaat agctaaggtc gaccgaggcc gcaggtaagt atcaaggtta
     4021 caagacaggt ttaaggagac cacatagaaa ctgggcttgt cgagacagag aagactcttg
     4081 cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt
     4141 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4201 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4261 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4321 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4381 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4441 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4501 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg
     4561 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4621 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4681 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga
     4741 attcgagctc ggtacgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     4801 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     4861 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     4921 tggggcagga cagcaagggg gaggattggg aagacaatag caggccagct tttgttccct
     4981 ttagggttaa ttgcgcgctt ggcgctaatc atggtcatag ctgtttccac tcctcaggtg
     5041 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     5101 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     5161 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     5221 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     5281 gtttagagtt tggcaacata tgccatatgc tggctgccat gaacaaaggt ggctataaag
     5341 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     5401 gacttgaggt tagatttttt tttatatttt gtttgtgtta tttttttctt taacatccct
     5461 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     5521 catagctgtc cctcttctct tatgaagatc cctcgacctg cagcccaagc ttggcgtaat
     5581 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5641 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5701 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
     5761 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     5821 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5881 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5941 aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     6001 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     6061 aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg
     6121 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     6181 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6241 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6301 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6361 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6421 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6481 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
     6541 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6601 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6661 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6721 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     6781 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6841 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6901 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6961 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7021 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7081 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7141 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7201 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7261 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7321 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7381 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7441 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7501 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7561 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7621 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7681 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7741 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7801 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7861 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7921 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7981 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8041 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
