LOCUS       sequence-176211-        6518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRB-tagged TagRFPT; targeted to plasma membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6518)
  AUTHORS   Mo GC, Ross B, Hertel F, Manna P, Yang X, Greenwald E, Booth C, 
            Plummer AM, Tenner B, Chen Z, Wang Y, Kennedy EJ, Cole PA, Fleming 
            KG, Palmer A, Jimenez R, Xiao J, Dedecker P, Zhang J
  TITLE     Genetically encoded biosensors for visualizing live-cell biochemical
            activity at super-resolution.
  JOURNAL   Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 
            2017 Mar 13.
  PUBMED    28288122
REFERENCE   2  (bases 1 to 6518)
  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: 
            "10.1038/nmeth.4221"; journalName: "Nat Methods"; date: "2017-04"; 
            volume: "14"; issue: "4"; pages: "427-434"
COMMENT     Sequence Label: FRB-TagRFPT-CAAX
FEATURES             Location/Qualifiers
     source          1..6518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             22..54
                     /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(58..66,67..81)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             88..366
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             394..1083
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGH"
     promoter        complement(1150..1168)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1151..1168)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1188..1205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1194..1418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1464..1892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1551..1570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1761..1782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1901..1921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1906..2235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2086..2221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2148..2167
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2302..3096
                     /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(2356..2375)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2966..2985
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3270..3391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3307..3326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3361..3380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3440..3456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3440..3456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3453..3475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3464..3480
                     /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(3488..3494,3495..3512,3513..3518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3533..3554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3671..3688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3842..4430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3922..3941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4601..5392,5393..5461))
                     /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     5224..5243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5462..5566)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5641..5660)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5832..6211
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6212..6415
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6366..6386
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6460..6479
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6460..6478
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      6498..6507
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat gggtcgggat
       61 ctgtacgacg atgacgataa ggatcccatc ctctggcatg agatgtggca tgaaggcctg
      121 gaagaggcat ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg
      181 gagcccttgc atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat
      241 caggcctatg gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca
      301 gggaatgtca aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc
      361 tcaaagacta gtggtaccgg aggtagcggt tctatgagcg agctgattaa ggagaacatg
      421 cacatgaagc tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag
      481 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
      541 cctctcccct tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc
      601 atcaaccaca cccagggcat ccccgatttc tttaagcagt ccttccctga gggcttcaca
      661 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
      721 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac
      781 ggccctgtga tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc
      841 gctgacggcg gcctggaagg cagaaccgac atggccctga agctcgtggg cgggggccac
      901 ctgatctgca acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg
      961 cccggcgtct actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc
     1021 tacgtcgagc agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg
     1081 cacaaggaat tcaagaagaa gaagaagagc aagaccaagt gcgtgatcat gtaatctaga
     1141 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     1201 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1261 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1321 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1381 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     1441 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1501 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1561 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1621 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1681 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1741 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1801 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1861 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     1921 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     1981 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2041 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2101 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2161 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2221 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2281 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2341 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2401 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2461 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     2521 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2581 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2641 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2701 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     2761 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     2821 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     2881 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     2941 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3001 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3061 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3121 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3181 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3241 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3301 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3361 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3421 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3481 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3541 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3601 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3661 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3721 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3781 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3841 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3901 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3961 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4021 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4081 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4141 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4201 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4261 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4321 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4381 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4441 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4501 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4561 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4621 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4681 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4741 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4801 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4861 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4921 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4981 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5041 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5101 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5161 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5221 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5281 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5341 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5401 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5461 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5521 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5581 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     5641 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     5701 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     5761 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     5821 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     5881 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5941 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     6001 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     6061 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     6121 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     6181 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     6241 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     6301 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     6361 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     6421 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     6481 gacccaagct tgcggccgcc accatggtgc ggggttct
//
