LOCUS       sequence-109040-        7481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cytosol-targeted, FRET-based PKA activity reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7481)
  AUTHORS   Herbst KJ, Allen MD, Zhang J
  TITLE     Spatiotemporally regulated protein kinase A activity is a critical 
            regulator of growth factor-stimulated extracellular signal-regulated
            kinase signaling in PC12 cells.
  JOURNAL   Mol Cell Biol. 2011 Oct;31(19):4063-75. doi: 10.1128/MCB.05459-11. 
            Epub 2011 Aug 1.
  PUBMED    21807900
REFERENCE   2  (bases 1 to 7481)
  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: 
            "10.1128/MCB.05459-11"; journalName: "Mol Cell Biol"; date: 
            "2011-10"; volume: "31"; issue: "19"; pages: "4063-75"
COMMENT     Sequence Label: pcDNA3-AKAR4-NES
FEATURES             Location/Qualifiers
     source          1..7481
                     /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"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /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(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1008..1010,1011..1013,1014..1724)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2247..2981
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     CDS             2988..3014
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     promoter        complement(3033..3051)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3034..3051)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3071..3088)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3077..3301
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3347..3775
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3434..3453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3644..3665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3784..3804)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3789..4119
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3970..4105
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4032..4051
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4186..4980
                     /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(4240..4259)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4850..4869
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5154..5275
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5191..5210)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5245..5264
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5324..5340)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5324..5340)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5337..5359)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5348..5364
                     /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(5372..5378,5379..5396,5397..5402))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5417..5438
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5555..5572)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5726..6314)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5806..5825)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6485..7276,7277..7345))
                     /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     7108..7127
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7346..7450)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatccatg gtgagcaagg
     1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1081 gccacaggtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1201 tgacctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgtac catcttcttc aaggacgacg
     1321 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1441 acgccatcag tgacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggccc
     1501 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1681 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcgcatg cataagtttt
     1741 ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg ggtcaaattc
     1801 ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga tccataaaga
     1861 aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac atttcaagac
     1921 tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta ttgaatgaca
     1981 tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc aatcagttac
     2041 tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt ttatctctgg
     2101 tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt gatcgctctg
     2161 caggtaagcc aggcagcggc gagggcagca ccaagggcct gcgtcgcgcc accctggtgg
     2221 acggcggcac cggcggcagc gagctcatgg gcggcgtgca gctcgccgac cactaccagc
     2281 agaacacccc catcggcgac ggccccgtgc tgctgccnga caaccactac ctgagctacc
     2341 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     2401 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaggt accggtggat
     2461 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2521 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2581 acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca
     2641 ccctcgtgac caccctgggc tacggccttc agtgcttcgc ccgctacccc gaccacatga
     2701 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2761 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2821 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2881 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     2941 acggcatcaa ggccaacttc aagatccgcc acaacatcga ggaattcctg ccccccctgg
     3001 agcgcctgac cctgtaatct agagggccct attctatagt gtcacctaaa tgctagagct
     3061 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3121 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3181 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3241 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3301 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3361 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3421 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3481 ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac
     3541 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3601 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3661 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg
     3721 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3781 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt
     3841 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3901 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3961 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4021 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4081 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4141 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4201 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4261 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4321 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4381 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4441 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4501 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4561 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4621 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4681 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4741 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4801 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4861 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4921 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4981 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5041 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5101 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5161 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5221 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5281 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5341 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5401 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5461 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5521 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5581 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5641 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5701 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5761 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5821 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5881 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     5941 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6001 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6061 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6121 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     6181 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6241 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6301 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6361 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6421 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6481 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6541 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6601 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6661 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6721 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6781 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6841 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6901 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6961 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7021 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7081 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7141 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7201 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7261 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7321 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7381 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7441 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
