LOCUS       sequence-362780-        7288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based sensor for monitoring Akt activity 
            near a protein of interest. Must pair with GFP11-tagged POI to 
            reconstitute donor fluorescence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7288)
  AUTHORS   Tenner B, Zhang JZ, Kwon Y, Pessino V, Feng S, Huang B, Mehta S, 
            Zhang J
  TITLE     FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized 
            signaling within endogenous microdomains.
  JOURNAL   Sci Adv. 2021 May 21;7(21). pii: 7/21/eabe4091. doi: 
            10.1126/sciadv.abe4091. Print 2021 May.
  PUBMED    34020947
REFERENCE   2  (bases 1 to 7288)
  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: "Sci Adv."; 
            date: "2021-05-21"; volume: "7(21). pii: 7/21/eabe4091. doi"; pages:
            " 10.1126/sciadv.abe4091. Print 2021 May"
COMMENT     Sequence Label: pcDNA3.1(+)-FluoSTEP-AktAR
FEATURES             Location/Qualifiers
     source          1..7288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        733..751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             789..1499
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RITVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREVAV
                     AKFAGLGGGMDELYK"
     CDS             2052..2693
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2749..2766)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2755..2979
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3025..3453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3112..3131)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3322..3343
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3462..3482)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3467..3796
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3647..3782
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3709..3728
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3863..4657
                     /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(3917..3936)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4527..4546
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4831..4952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4868..4887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4922..4941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5001..5017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5001..5017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5014..5036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5025..5041
                     /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(5049..5055,5056..5073,5074..5079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5094..5115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5232..5249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5403..5991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5483..5502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6162..6953,6954..7022))
                     /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     6785..6804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7023..7127)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7202..7221)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      721 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
      781 aagcttggat ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg
      841 tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc
      901 cgtacatggg aactcaaacc atgaggatca cagtcatcga gggaggaccc ctgccatttg
      961 cctttgacat tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga
     1021 aaggcattcc tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta
     1081 cgagatacga agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct
     1141 gtctcgttta ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc
     1201 agaagaagac caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc
     1261 tgaggggata cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt
     1321 tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg
     1381 ccgttgatca ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac
     1441 gcgaagtcgc agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagc
     1501 gcatgcataa gttttctcaa gaacagatcg gcgaaaacat tgtgtgcagg gtcatttgta
     1561 ccacgggtca aattcccatc cgagatttgt cagctgatat ttcacaagtg cttaaggaaa
     1621 aacgatccat aaagaaagtt tggacatttg gtagaaaccc agcctgtgac tatcatttag
     1681 gaaacatttc aagactgtca aataagcatt tccaaatact actaggagaa gacggtaacc
     1741 ttttattgaa tgacatttcc actaatggga cctggttaaa tgggcaaaaa gtcgagaaga
     1801 acagcaatca gttactgtct caaggtgatg aaataaccgt tggtgtaggc gtggaatcag
     1861 atattttatc tctggtcatt ttcataaacg acaaatttaa gcagtgcctc gagcagaaca
     1921 aagttgatcg ctctgcaggt aagccaggca gcggcgaggg cagcaccaag ggcctggtcg
     1981 accctcgtcc gcgctcgtgc acctggccgg accccaggcc ggagtttgga ggtaccggcg
     2041 gcagtgaact tatgtccaaa ggagaagaac tgtttaccgg tgttgtgcca attttggttg
     2101 aactcgatgg tgatgtcaac ggacataagt tctcagtgag aggcgaagga gaaggtgacg
     2161 ccaccattgg aaaattgact cttaaattca tctgtactac tggtaaactt cctgtaccat
     2221 ggccgactct cgtaacaacg cttacgtacg gagttcagtg cttttcgaga tacccagacc
     2281 atatgaaaag acatgacttt tttaagtcgg ctatgcctga aggttacgtg caagaaagaa
     2341 caatttcgtt caaagatgat ggaaaatata aaactagagc agttgttaaa tttgaaggag
     2401 atactttggt taaccgcatt gaactgaaag gaacagattt taaagaagat ggtaatattc
     2461 ttggacacaa actcgaatac aattttaata gtcataacgt atacatcact gctgataagc
     2521 aaaagaacgg aattaaagcg aatttcacag tacgccataa tgtagaagat ggcagtgttc
     2581 aacttgccga ccattaccaa caaaacaccc ctattggaga cggtccggta cttcttcctg
     2641 ataatcacta cctctcaaca caaacagtcc tgagcaaaga tccaaatgaa aaaggaacag
     2701 gtggcggcgg aagttagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     2761 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     2821 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     2881 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2941 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3001 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3061 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3121 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3181 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3241 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3301 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3361 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3421 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3481 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3541 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3601 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3661 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3721 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3781 cctaggcttt tgcaaaaagc tcccgggagc ttatatatcc attttcggat ctgatcaaga
     3841 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3901 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3961 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4021 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4081 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4141 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4201 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4261 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4321 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4381 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4441 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4501 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4561 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4621 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4681 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4741 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4801 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4861 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4921 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4981 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5041 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5101 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5221 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5281 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5341 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5401 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5461 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5521 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5581 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5641 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5701 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5761 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5821 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5881 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5941 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6001 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6061 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6121 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6181 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6241 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6301 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6361 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6421 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6481 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6541 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6601 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6661 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6721 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6781 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6841 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6901 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6961 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7021 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7141 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7201 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7261 aggtcgctga gtagtgcgcg agcaaaat
//
