LOCUS       sequence-414096-        6665 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses plasma-membrane-bound FRB-SBFP2 for rapid relocalization 
            of cytoplasmic proteins tagged with FKBP upon addition of rapamycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6665)
  AUTHORS   Wordeman L, Decarreau J, Vicente JJ, Wagenbach M
  TITLE     Divergent microtubule assembly rates after short- versus long-term 
            loss of end-modulating kinesins.
  JOURNAL   Mol Biol Cell. 2016 Apr 15;27(8):1300-9. doi: 
            10.1091/mbc.E15-11-0803. Epub 2016 Feb 24.
  PUBMED    26912793
REFERENCE   2  (bases 1 to 6665)
  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"; doi: 
            "10.1091/mbc.E15-11-0803"; journalName: "Mol Biol Cell"; date: 
            "2016-04-15- 15"; volume: "27"; issue: "8"; pages: "1300-9"
COMMENT     Sequence Label: pMX1463
FEATURES             Location/Qualifiers
     source          1..6665
                     /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"
     5'UTR           963..1002
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     regulatory      1017..1026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1095..1373
                     /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             join(1386..1388,1389..1391,1392..2102)
                     /codon_start=1
                     /product="strongly enhanced blue fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1431..1452)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1692..1711)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2039..2060
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2103..2168
                     /label=MCS
                     /note="multiple cloning site"
     promoter        complement(2218..2236)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2219..2236)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2256..2273)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2262..2486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2532..2960
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2619..2638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2829..2850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2969..2989)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2974..3303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3154..3289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3216..3235
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3370..4164
                     /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(3424..3443)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4034..4053
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4338..4459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4375..4394)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4429..4448
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4508..4524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4508..4524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4521..4543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4532..4548
                     /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(4556..4562,4563..4580,4581..4586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4601..4622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4739..4756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4910..5498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4990..5009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5669..6460,6461..6529))
                     /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     6292..6311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6530..6634)
                     /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 gcttggtacc
      901 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      961 gggatcttgg tggcgtgaaa ctcccgcacc tcttcggcca gcgaattcca gaagccgcca
     1021 ccatgggatg tataaaatca aaagggaaag acagcgcggg agcagatagt gctggtagtg
     1081 ctggtagtgc tggtatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc
     1141 gtttgtactt tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg
     1201 ctatgatgga acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc
     1261 gagatttaat ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg
     1321 acctcctcca agcctgggac ctctattatc atgtgttccg acgaatctca aagactagtt
     1381 atccgatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1441 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1501 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1561 ccaccctcgt gaccaccctg agccacggcg tgcagtgctt cgcccgctac cccgaccaca
     1621 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1681 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1741 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1801 ggcacaagct ggagtacaac ttcaacagcc acaacgtcta tatcaccgcc gacaagcaga
     1861 agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc
     1921 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1981 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     2041 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2101 agtccggact cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc
     2161 cgggatccac cggatctaga taagcggccg ctcgagcatg catctagagg gccctattct
     2221 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     2281 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2341 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2401 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2461 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2521 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2581 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2641 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2701 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2761 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2821 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2881 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2941 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3001 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3121 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3181 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3241 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3301 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     3361 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3421 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3481 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3541 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3601 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3661 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3721 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3781 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3841 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     3901 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     3961 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4021 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4081 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4141 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4201 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4261 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4321 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4381 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4441 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4501 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4561 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4621 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4681 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4741 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4801 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4861 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4921 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4981 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5041 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5101 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5161 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5221 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5281 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5341 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5401 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5461 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5521 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5581 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5641 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5701 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5761 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5821 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5881 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5941 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6001 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6061 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6121 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6181 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6241 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6301 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6361 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6421 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6481 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6541 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6601 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6661 acgtc
//
