LOCUS       sequence-345236-        7481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yellow-red FRET-based Akt/PKB activity reporter for multiplexed 
            imaging applications..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7481)
  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 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"; journalName: "Nat 
            Commun."; date: "2020-04-15"; volume: "11(1):1848. doi"; pages: " 
            10.1038/s41467-020-15687-x"
COMMENT     Sequence Label: pcDNA3-Venus-StagRFP-AktAR
FEATURES             Location/Qualifiers
     source          1..7481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..59)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        231..610
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..814
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     765..785
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     859..878
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        859..877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      897..906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             918..935
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             939..971
                     /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(975..983,984..998)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1005..1007,1008..1010,1011..1721)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1050..1071)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1311..1330)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1658..1679
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2274..2969
                     /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(3030..3048)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3031..3048)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3068..3085)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3074..3298
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3344..3772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3431..3450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3641..3662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3781..3801)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3786..4115
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3966..4101
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4028..4047
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4182..4976
                     /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(4236..4255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4846..4865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5150..5271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5187..5206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5241..5260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5320..5336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5320..5336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5333..5355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5344..5360
                     /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(5368..5374,5375..5392,5393..5398))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5413..5434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5551..5568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5722..6310)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5802..5821)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6481..7272,7273..7341))
                     /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     7104..7123
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7342..7446)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
       61 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
      121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      241 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      301 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      361 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      421 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      481 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      541 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      601 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      661 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      721 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      781 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
      841 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt gcggccgcca
      901 ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg actggtggac
      961 agcaaatggg tcgggatctg tacgacgatg acgataagga tcccatggtg agcaagggcg
     1021 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1081 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1141 agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg
     1201 gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca
     1261 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1321 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1381 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1441 acaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact
     1501 tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga
     1561 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt
     1621 ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1681 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gcgcatgcat aagttttctc
     1741 aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt caaattccca
     1801 tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc ataaagaaag
     1861 tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt tcaagactgt
     1921 caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg aatgacattt
     1981 ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat cagttactgt
     2041 ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta tctctggtca
     2101 ttttcataaa cgacaaattt aagcagtgcc tcgagcagaa caaagttgat cgctctgcag
     2161 gtaagccagg cagcggcgag ggcagcacca agggcctggt cgaccctcgt ccgcgctcgt
     2221 gcacctggcc ggaccccagg ccggagtttg gaggtaccgg cggcagcgag ctcatgagcg
     2281 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc
     2341 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     2401 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca
     2461 tgtacggcag cagaaccttc atcaaccaca cccagggcat ccccgatttc tttaagcagt
     2521 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     2581 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     2641 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcca
     2701 acaccgagat gctgtacccc gctgacggcg gcctggaagg cagaaccgtc atggccctga
     2761 agctcgtggg cgggggccac ctgatctgca acttcaagac cacatacaga tccaagaaac
     2821 ccgctaagaa cctcaagatg cccggcgtct actatgtgga ccacagactg gaaagaatca
     2881 aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg
     2941 acctccctag caaactgggg cacaagtaag aattctgcag atatccatca cactggcggc
     3001 cgctcgagca tgcatctaga gggccctatt ctatagtgtc acctaaatgc tagagctcgc
     3061 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3121 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3181 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3241 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3301 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3361 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3421 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3481 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3541 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3601 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3661 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3721 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3781 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3841 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3901 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3961 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4021 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4081 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4141 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     4201 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     4261 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4321 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     4381 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4441 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4501 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4561 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4621 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4681 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     4741 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4801 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4861 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4921 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4981 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     5041 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     5101 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     5161 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     5221 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     5281 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5341 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5401 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5461 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5521 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5581 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5641 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5701 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5761 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5821 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5881 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5941 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6001 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6061 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6121 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6181 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6241 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6301 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6361 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6421 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6481 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6541 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6601 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6661 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6721 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6781 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6841 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6901 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6961 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7021 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7081 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7141 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7201 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7261 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7321 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7381 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7441 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac g
//
