LOCUS       sequence-298096-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET-based biosensor for monitoring PKA activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  AUTHORS   Depry C, Allen MD, Zhang J
  TITLE     Visualization of PKA activity in plasma membrane microdomains.
  JOURNAL   Mol Biosyst. 2011 Jan;7(1):52-8. doi: 10.1039/c0mb00079e. Epub 2010 
            Sep 14.
  PUBMED    20838685
REFERENCE   2  (bases 1 to 7493)
  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: 
            "10.1039/c0mb00079e"; journalName: "Mol Biosyst"; date: "2011-01"; 
            volume: "7"; issue: "1"; pages: "52-8"
COMMENT     Sequence Label: pcDNA3-AKAR4
FEATURES             Location/Qualifiers
     source          1..7493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             709..741
                     /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(745..753,754..768)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(775..777,778..780,781..1491)
                     /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             2014..2748
                     /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"
     promoter        complement(2812..2830)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2813..2830)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2850..2867)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2856..3080
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3126..3554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3213..3232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3423..3444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3563..3583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3568..3897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3748..3883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3810..3829
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3964..4758
                     /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(4018..4037)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4628..4647
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4932..5053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4969..4988)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5023..5042
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5102..5118)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5102..5118)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5115..5137)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5126..5142
                     /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(5150..5156,5157..5174,5175..5180))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5195..5216
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5333..5350)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5504..6092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5584..5603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6263..7054,7055..7123))
                     /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     6886..6905
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7124..7228)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7303..7322)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gcggccgcca ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg
      721 actggtggac agcaaatggg tcgggatctg tacgacgatg acgataagga tcccatggtg
      781 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
      841 gtaaacggcc acaggttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
      901 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
      961 accaccctga cctggggcgt gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgtaccat cttcttcaag
     1081 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1201 gagtacaacg ccatcagtga caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1261 aaggcccact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1321 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1381 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1441 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gcgcatgcat
     1501 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt
     1561 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc
     1621 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt
     1681 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg
     1741 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat
     1801 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta
     1861 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgagcagaa caaagttgat
     1921 cgctctgcag gtaagccagg cagcggcgag ggcagcacca agggcctgcg tcgcgccacc
     1981 ctggtggacg gcggcaccgg cggcagcgag ctcatgggcg gcgtgcagct cgccgaccac
     2041 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2101 agctaccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2161 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggaggtacc
     2221 ggtggatcta tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2281 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2341 gccacctacg gcaagctgac cctgaagctg atctgcacca ccggcaagct gcccgtgccc
     2401 tggcccaccc tcgtgaccac cctgggctac ggccttcagt gcttcgcccg ctaccccgac
     2461 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2521 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2581 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2641 ctggggcaca agctggagta caactacaac agccacaacg tctatatcac cgccgacaag
     2701 cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagta agaattctgc
     2761 agatatccat cacactggcg gccgctcgag catgcatcta gagggcccta ttctatagtg
     2821 tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2881 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2941 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3001 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3061 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3121 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3181 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3241 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3301 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3361 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3421 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3481 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3541 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3601 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3661 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3721 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3781 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3841 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3901 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3961 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     4021 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     4081 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     4141 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     4201 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     4261 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     4321 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     4381 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     4441 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     4501 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     4561 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4621 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4681 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4741 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4801 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4861 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4921 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4981 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5041 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     5101 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5161 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     5221 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5281 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5341 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5401 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5461 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5521 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5581 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5641 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5701 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5761 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5821 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5881 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5941 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6001 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6061 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6121 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6181 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6241 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6301 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6361 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6421 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6481 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6541 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6601 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6661 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6721 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6781 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6841 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6901 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6961 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7021 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7081 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7141 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7201 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     7261 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     7321 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     7381 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     7441 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
