LOCUS       sequence-118087-        5160 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Mitochondria 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 5160)
  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 5160)
  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: ECFP(W66A)-FRB-MoA
FEATURES             Location/Qualifiers
     source          1..5160
                     /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      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTAGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1342..1620
                     /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"
     polyA_signal    1948..2069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1985..2004)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2039..2058
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2076..2531)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2213..2234)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2425..2444
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2558..2662
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2664..3021
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2664..2855
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2687..2707)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2872..3007
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3056..3850
                     /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             3056..3850
                     /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(3110..3129)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3720..3739
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4038..4057)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4082..4129
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4458..5046
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4947..4966
                     /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 gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc gccggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac atcagccaca acgtctatat caccgccgac
     1081 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagg gagcaagtgg aatcctctgg catgagatgt ggcatgaagg cctggaagag
     1381 gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc
     1441 ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc
     1501 tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat
     1561 gtcaaggacc tcctccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag
     1621 agtgctggtg gtagtgctgg tggtagtgct ggtggtactg gtggtcctcg agctcaagct
     1681 tcgaattcga gtgctggtgg tttctgggaa aggaacctgc cctctgtttc tggcctgctg
     1741 aagatcattg gattttccac atcagtaact gccctggggt ttgtgctgta caaatacaag
     1801 ctcctgccac ggtcttgagg atccaccgga tctagataac tgatcataat cagccatacc
     1861 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     1921 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     1981 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2041 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     2101 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2161 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2221 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2281 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     2341 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     2401 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     2461 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     2521 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2581 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2641 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     2701 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2761 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2821 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2881 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2941 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3001 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3061 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3121 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3181 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     3241 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     3301 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     3361 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     3421 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     3481 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     3541 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     3601 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     3661 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     3721 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     3781 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     3841 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     3901 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     3961 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4021 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4081 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     4141 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     4201 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     4261 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     4321 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4381 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4441 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4501 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4561 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4621 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4681 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4741 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4801 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4861 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4921 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4981 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5041 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5101 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
