LOCUS       sequence-205450-        6602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Frb-EGFP-HOTag6 fusion in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6602)
  AUTHORS   Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis 
            A, Coughlin SR, Kornberg TB, Shu X
  TITLE     Visualizing Dynamics of Cell Signaling In Vivo with a Phase 
            Separation-Based Kinase Reporter.
  JOURNAL   Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008.
  PUBMED    29307513
REFERENCE   2  (bases 1 to 6602)
  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: "Mol Cell. 
            2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 
            10.1016/j.molcel.2017.12.008."
COMMENT     Sequence Label: pcDNA3-Frb-EGFP-HOTag6
FEATURES             Location/Qualifiers
     source          1..6602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        145..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        525..728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     679..699
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     773..792
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        773..791
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      833..842
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             842..1114
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQL"
     CDS             join(1154..1156,1157..1159,1160..1870)
                     /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(1199..1220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1460..1479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1807..1828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2103..2120)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2109..2333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2379..2807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2466..2485)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2676..2697
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2816..2836)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2821..3150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3001..3136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3063..3082
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3217..4011
                     /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(3271..3290)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3881..3900
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4185..4306
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4222..4241)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4276..4295
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4355..4371)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4355..4371)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4368..4390)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4379..4395
                     /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(4403..4409,4410..4427,4428..4433))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4448..4469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4586..4603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4757..5345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4837..4856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5516..6307,6308..6376))
                     /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     6139..6158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6377..6481)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6556..6575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
       61 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      121 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      601 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      661 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      721 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      781 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttggta ccgccaccat
      841 ggagatgtgg cacgagggac tcgaagaggc cagcaggctg tactttggcg agaggaacgt
      901 caagggcatg ttcgaagtgc tggagcccct ccatgcgatg atggaaaggg gcccacagac
      961 cctgaaggag accagcttca accaggctta cggcagggac ctgatggagg cacaggaatg
     1021 gtgcaggaag tacatgaaga gcggcaacgt gaaagacctg acccaggcgt gggacctcta
     1081 ctaccacgtg ttcaggagga tcagcaagca gctcgagtcc ggactcagat ctggcagcgg
     1141 tggaggcgat atcatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1201 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1261 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1321 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1381 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1441 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1501 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1561 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1621 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag
     1681 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1741 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1801 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1861 gctgtacaag ggatccggca gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc
     1921 tggtggcagt gctggtggta gtgctggtgg tagtgctggt ggctctagaa ccttgagaga
     1981 aatcgaggaa ttgttgagga aaatcattga agacagcgtg cgcagtgtgg ccgagctgga
     2041 ggacattgag aagtggctga agaaaatcta agaattcggg cccgtttaaa cccgctgatc
     2101 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2161 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2221 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2281 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2341 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2401 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2461 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2521 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2581 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2641 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2701 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2761 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2821 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2881 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2941 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3001 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3061 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3121 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3181 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3241 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3301 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3361 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3421 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3481 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3541 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3601 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3661 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3721 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3781 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3841 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3901 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3961 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4021 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4081 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4141 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4201 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4261 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4321 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4381 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4441 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6361 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6481 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     6541 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     6601 tg
//
