LOCUS       sequence-100421-        6543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lipid raft-targeted FRET biosensor for monitoring PKA activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6543)
  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 6543)
  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-Lyn-AKAR4
FEATURES             Location/Qualifiers
     source          1..6543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      13..22
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(58..60,61..63,64..774)
                     /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             1297..2031
                     /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(2095..2113)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2096..2113)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2133..2150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2139..2363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2409..2837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2496..2515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2706..2727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2846..2866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2851..3181
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3032..3167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3094..3113
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3248..4042
                     /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(3302..3321)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3912..3931
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4216..4337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4253..4272)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4307..4326
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4386..4402)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4386..4402)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4399..4421)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4410..4426
                     /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(4434..4440,4441..4458,4459..4464))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4479..4500
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4617..4634)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4788..5376)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4868..4887)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5547..6338,6339..6407))
                     /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     6170..6189
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6408..6512)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aagcttgcgg ccgccaccat gggctgcatc aagagcaagc gcaaggacaa ggatcccatg
       61 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      121 gacgtaaacg gccacaggtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      181 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      241 gtgaccaccc tgacctgggg cgtgcagtgc ttcgcccgct accccgacca catgaagcag
      301 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgtac catcttcttc
      361 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      421 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      481 ctggagtaca acgccatcag tgacaacgtc tatatcaccg ccgacaagca gaagaacggc
      541 atcaaggccc acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      601 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      661 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      721 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagcgcatg
      781 cataagtttt ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg
      841 ggtcaaattc ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga
      901 tccataaaga aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac
      961 atttcaagac tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta
     1021 ttgaatgaca tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc
     1081 aatcagttac tgtctcaagg tgatgaaata accgttggtg taggcgtgga atcagatatt
     1141 ttatctctgg tcattttcat aaacgacaaa tttaagcagt gcctcgagca gaacaaagtt
     1201 gatcgctctg caggtaagcc aggcagcggc gagggcagca ccaagggcct gcgtcgcgcc
     1261 accctggtgg acggcggcac cggcggcagc gagctcatgg gcggcgtgca gctcgccgac
     1321 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgccnga caaccactac
     1381 ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1441 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaggt
     1501 accggtggat ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1561 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1621 gatgccacct acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg
     1681 ccctggccca ccctcgtgac caccctgggc tacggccttc agtgcttcgc ccgctacccc
     1741 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1801 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1861 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1921 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac
     1981 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga gtaagaattc
     2041 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata
     2101 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     2161 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2221 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2281 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2341 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2401 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2461 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2521 tctcgccacg ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt
     2581 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2641 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2701 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2761 tgatttataa gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca
     2821 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2881 ggctccccag gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2941 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3001 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3061 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3121 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3181 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     3241 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3301 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3361 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     3421 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     3481 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     3541 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     3601 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     3661 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     3721 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     3781 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     3841 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     3901 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     3961 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4021 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4081 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4141 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4201 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4261 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4321 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     4381 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4441 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4501 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4561 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4621 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4681 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4741 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4801 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4861 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4921 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4981 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5041 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5101 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5161 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5221 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5281 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5341 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5401 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5461 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5521 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5581 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5641 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5701 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5761 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5821 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5881 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5941 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6001 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6061 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6121 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6181 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6241 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6301 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6361 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6421 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6481 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6541 gtc
//
