LOCUS       sequence-118088-        5468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Golgi 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 5468)
  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 5468)
  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: FRB-ECFP(W66A)-Giantin
FEATURES             Location/Qualifiers
     source          1..5468
                     /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"
     regulatory      600..609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             618..896
                     /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             903..929
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      993..1002
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(999..1001,1002..1004,1005..1715)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTAGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1044..1065)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1305..1324)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1652..1673
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2256..2377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2293..2312)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2347..2366
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2384..2839)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2521..2542)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2733..2752
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2866..2970
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2972..3329
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2972..3163
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2995..3015)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3180..3315
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3364..4158
                     /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             3364..4158
                     /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(3418..3437)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4028..4047
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4346..4365)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4390..4437
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4766..5354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5255..5274
                     /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 gctagcgccg
      601 ccaccatggc ttctagaatc ctctggcatg agatgtggca tgaaggcctg gaagaggcat
      661 ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc
      721 atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg
      781 gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtca
      841 aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagacta
      901 gttatccgta cgacgtacca gactacgcaa gtgctggtag tgctggtagt gctggtagtg
      961 ctggtagtgc tggtagtgct ggttcaccgg tcgccaccat ggtgagcaag ggcgaggagc
     1021 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1081 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1141 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgaccgccg
     1201 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1261 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1321 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1381 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacatca
     1441 gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga
     1501 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1561 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     1621 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1681 ccgggatcac tctcggcatg gacgagctgt acaagtccgg actcagatct cgaggagaac
     1741 cgcagcaaag cttttctgaa gctcagcagc agctatgcaa caccagacag gaagtgaatg
     1801 aattaaggaa gctgctggaa gaagaacgag accaaagagt ggctgctgag aatgctctct
     1861 ctgtggccga ggagcagatc agacggttag agcacagtga atgggactct tcccggactc
     1921 ctatcattgg ctcctgtggc actcaggagc aggcactgtt aatagatctt acaagcaaca
     1981 gttgtcgaag gacccggagt ggcgttggat ggaagcgagt cctgcgttca ctctgtcatt
     2041 cacggacccg agtgccactt ctagcagcca tctactttct aatgattcat gtcctgctca
     2101 ttctgtgttt tacgggccat ctatagggat ccaccggatc tagataactg atcataatca
     2161 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2221 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     2281 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2341 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat
     2401 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2461 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2521 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2581 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2641 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2701 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2761 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2821 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2941 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     3001 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3061 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3121 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3181 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3241 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3301 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     3361 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3421 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3481 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3541 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3601 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3661 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3721 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3781 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3841 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     3901 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3961 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     4021 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     4081 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4141 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4201 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4261 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4321 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     4381 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     4441 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     4501 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     4561 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     4621 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4681 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4741 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4801 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4861 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4921 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4981 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5041 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5101 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5161 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5221 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5281 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5341 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5401 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5461 ccatgcat
//
