LOCUS       sequence-118089-        5059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a membrane located FRB domain in mammalian cells (the ECFP
            is not fluorescent).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5059)
  AUTHORS   van Unen J, Reinhard NR, Yin T, Wu YI, Postma M, Gadella TW, 
            Goedhart J
  TITLE     Plasma membrane restricted RhoGEF activity is sufficient for 
            RhoA-mediated actin polymerization.
  JOURNAL   Sci Rep. 2015 Oct 5;5:14693. doi: 10.1038/srep14693.
  PUBMED    26435194
REFERENCE   2  (bases 1 to 5059)
  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 Rep."; 
            date: "2015-10-5"; volume: "5:14693. doi"; pages: " 
            10.1038/srep14693"
COMMENT     Sequence Label: Lck-FRB-ECFP(W66A)
FEATURES             Location/Qualifiers
     source          1..5059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             complement(649..666)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             681..959
                     /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             966..992
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1008..1010,1011..1724)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTAGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     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"
     polyA_signal    1847..1968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1884..1903)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1938..1957
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1975..2430)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2112..2133)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2324..2343
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2457..2561
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2563..2920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2563..2754
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2586..2606)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2771..2906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2955..3749
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2955..3749
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3009..3028)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3619..3638
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3937..3956)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3981..4028
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4357..4945
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4846..4865
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacca
      601 tgggctgcgt gtgcagcagc aaccccgaga gatctgcgaa ttcaggtgct ggtgctggtg
      661 ctggtgctat cctatctaga atcctctggc atgagatgtg gcatgaaggc ctggaagagg
      721 catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct
      781 tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt aatcaggcct
      841 atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg
      901 tcaaggacct cctgcaagcc tgggacctct attatcatgt gttccgacga atctcaaaga
      961 ctagttatcc gtacgacgta ccagactacg caccggtcgc caccctggtg agcaagggcg
     1021 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1081 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1141 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1201 ccgccggcgt gcagtgcttc agccgctacc 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 acatcagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact
     1501 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1561 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1621 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1681 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta
     1741 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     1801 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     1861 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     1921 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg
     1981 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2041 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2101 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2161 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2221 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2281 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2341 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2401 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     2461 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2521 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     2581 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2641 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2701 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2761 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2821 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2881 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     2941 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3001 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3061 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3121 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3181 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3241 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3301 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3361 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3421 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3481 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3541 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3601 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3661 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3721 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3781 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3841 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3901 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     3961 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     4021 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     4081 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     4141 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     4201 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4261 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4321 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4381 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4441 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4501 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4561 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4621 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4681 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4741 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4801 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4861 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4921 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4981 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5041 ccgtattacc gccatgcat
//
