LOCUS       sequence-201712-        6593 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 6593)
  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 6593)
  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..6593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             926..1198
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQL"
     CDS             join(1238..1240,1241..1243,1244..1954)
                     /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(1283..1304)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1544..1563)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1891..1912
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2187..2204)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2193..2417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2463..2891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2550..2569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2760..2781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2900..2920)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2905..3234
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3085..3220
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3147..3166
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3301..4095
                     /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(3355..3374)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3965..3984
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4269..4390
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4306..4325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4360..4379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4439..4455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4439..4455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4452..4474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4463..4479
                     /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(4487..4493,4494..4511,4512..4517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4532..4553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4670..4687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4841..5426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4921..4940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5597..6388,6389..6457))
                     /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     6220..6239
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6458..6562)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttgcca ccatggagat gtggcacgag ggactcgaag aggccagcag
      961 gctgtacttt ggcgagagga acgtcaaggg catgttcgaa gtgctggagc ccctccatgc
     1021 gatgatggaa aggggcccac agaccctgaa ggagaccagc ttcaaccagg cttacggcag
     1081 ggacctgatg gaggcacagg aatggtgcag gaagtacatg aagagcggca acgtgaaaga
     1141 cctgacccag gcgtgggacc tctactacca cgtgttcagg aggatcagca agcagctcga
     1201 gtccggactc agatctggca gcggtggagg cgatatcatg gtgagcaagg gcgaggagct
     1261 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1321 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1381 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1441 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1501 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1561 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1621 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1681 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     1741 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1801 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1861 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1921 cgggatcact ctcggcatgg acgagctgta caagggatcc ggcagtgctg gtggtagtgc
     1981 tggtggtagt gctggtggta gtgctggtgg cagtgctggt ggtagtgctg gtggtagtgc
     2041 tggtggctct agaaccttga gagaaatcga ggaattgttg aggaaaatca ttgaagacag
     2101 cgtgcgcagt gtggccgagc tggaggacat tgagaagtgg ctgaagaaaa tctaagaatt
     2161 cgggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2221 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2281 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2341 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2401 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2461 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2941 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3001 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3061 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3121 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3181 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3241 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3301 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3361 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3421 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3481 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3541 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3601 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3661 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3721 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3781 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3841 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3901 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3961 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4021 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4081 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4141 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4201 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4261 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4321 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4381 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5341 gctcttgatc cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga
     5401 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5461 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5521 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5581 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5641 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5701 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5761 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5821 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5881 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5941 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6001 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6061 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6121 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6181 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6241 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6301 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6361 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6421 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6481 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6541 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
