LOCUS       sequence-345233-        7670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasma membrane-targeted variant of improved green-red FRET-based 
            PKA activity reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7670)
  AUTHORS   Mo GCH, Posner C, Rodriguez EA, Sun T, Zhang J
  TITLE     A rationally enhanced red fluorescent protein expands the utility of
            FRET biosensors.
  JOURNAL   Nat Commun. 2020 Apr 15;11(1):1848. doi: 10.1038/s41467-020-15687-x.
  PUBMED    32296061
REFERENCE   2  (bases 1 to 7670)
  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"; journalName: "Nat 
            Commun."; date: "2020-04-15"; volume: "11(1):1848. doi"; pages: " 
            10.1038/s41467-020-15687-x"
COMMENT     Sequence Label: pcDNA3-Lyn-GR-AKAR3
FEATURES             Location/Qualifiers
     source          1..7670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(71..90)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        262..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     890..909
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        890..908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      928..937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(973..975,976..978,979..1689)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1018..1039)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1279..1298)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1626..1647
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2494..3189
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTVMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     promoter        complement(3250..3268)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3251..3268)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3288..3305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3564..3992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3651..3670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3861..3882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4001..4021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4006..4335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4186..4321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4248..4267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4402..5196
                     /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(4456..4475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5066..5085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5370..5491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5407..5426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5461..5480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5540..5556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5540..5556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5553..5575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5564..5580
                     /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(5588..5594,5595..5612,5613..5618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5633..5654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5771..5788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5942..6530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6022..6041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6701..7492,7493..7561))
                     /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     7324..7343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7562..7666)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
       61 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      121 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      181 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      241 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      781 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      901 actataggga gacccaagct tgcggccgcc accatgggct gcatcaagag caagcgcaag
      961 gacaaggatc ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1021 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1081 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1141 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1201 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1261 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1321 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1381 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1441 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcagc
     1501 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1561 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1621 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1681 ctgtacaaga gatctatgca taagttttct caagaacaga tcggcgaaaa cattgtgtgc
     1741 agggtcattt gtaccacggg tcaaattccc atccgagatt tgtcagctga tatttcacaa
     1801 gtgcttaagg aaaaacgatc cataaagaaa gtttggacat ttggtagaaa cccagcctgt
     1861 gactatcatt taggaaacat ttcaagactg tcaaataagc atttccaaat actactagga
     1921 gaagacggta accttttatt gaatgacatt tccactaatg ggacctggtt aaatgggcaa
     1981 aaagtcgaga agaacagcaa tcagttactg tcccaaggtg atgaaataac cgttggtgta
     2041 ggcgtggaat cagatatttt atctctggtc attttcataa acgacaaatt taagcagtgc
     2101 ctggagcaga acaaagttga tcgcggtacc agtgctggtg gtagtgctgg tggtagtgct
     2161 ggtggtagtg ctggtggtag tgctggtggt tccggcagtg ctggtggtag tgctggtggt
     2221 agtaccagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct
     2281 ggtggttccg gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg tggtagtgct
     2341 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt
     2401 agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg ctggtggttc cggattgagg
     2461 cgcgcgacgc tggttgacgg cggcagcgag ctcatgagcg agctgattaa ggagaacatg
     2521 cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag
     2581 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     2641 cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc
     2701 atcaaccaca cccagggcat ccccgatttc tttaagcagt ccttccctga gggcttcaca
     2761 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     2821 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac
     2881 ggccctgtga tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc
     2941 gctgacggcg gcctggaagg cagaaccgtc atggccctga agctcgtggg cgggggccac
     3001 ctgatctgca acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg
     3061 cccggcgtct actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc
     3121 tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg
     3181 cacaagtaag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     3241 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     3301 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3361 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3421 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3481 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3541 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3601 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3661 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3721 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3781 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3841 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3901 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3961 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4021 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4081 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4141 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4201 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4261 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4321 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4381 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4441 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4501 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4561 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4621 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4681 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4741 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4801 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4861 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4921 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4981 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5041 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5101 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5161 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5221 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5281 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5341 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5401 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5461 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5521 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5581 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5641 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5701 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5761 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5821 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6361 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7141 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7201 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7261 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7321 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7381 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7441 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7501 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7561 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7621 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
//
