LOCUS       sequence-176112-        6329 bp    DNA     circular SYN 15-APR-2025
DEFINITION  FRB-tagged N-terminal Dronpa fragment for BiFC; localizes to actin 
            cytoskeleton..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6329)
  AUTHORS   Hertel F, Mo GC, Duwe S, Dedecker P, Zhang J
  TITLE     RefSOFI for Mapping Nanoscale Organization of Protein-Protein 
            Interactions in Living Cells.
  JOURNAL   Cell Rep. 2016 Jan 12;14(2):390-400. doi: 
            10.1016/j.celrep.2015.12.036. Epub 2015 Dec 31.
  PUBMED    26748717
REFERENCE   2  (bases 1 to 6329)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2015.12.036"; journalName: "Cell Rep"; date: 
            "2016-01-12- 12"; volume: "14"; issue: "2"; pages: "390-400"
COMMENT     Sequence Label: pcDNA3-LifeAct-FRB-DMVF-181-N
FEATURES             Location/Qualifiers
     source          1..6329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..51
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             61..339
                     /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"
     promoter        complement(979..997)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(980..997)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1017..1034)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1023..1247
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1293..1721
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1380..1399)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1590..1611
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1730..1750)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1735..2064
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1915..2050
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1977..1996
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2131..2925
                     /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(2185..2204)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2795..2814
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3099..3220
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3136..3155)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3190..3209
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3269..3285)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3269..3285)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3282..3304)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3293..3309
                     /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(3317..3323,3324..3341,3342..3347))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3362..3383
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3500..3517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3671..4259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3751..3770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4430..5221,5222..5290))
                     /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     5053..5072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5291..5395)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5470..5489)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5661..6040
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6041..6244
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6195..6215
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6289..6308
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6289..6307
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(6324..6329,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atgggcgtgg ccgacctgat caagaagttc gagagcatca gcaaggagga gggggatccc
       61 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg
      121 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg
      181 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag
      241 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc
      301 tgggacctct attatcatgt gttccgacga atctcaaaga ctagtggtac cggtggaggt
      361 ggatctggag gaatgagtgt gattaaacca gacatgaaga tcaagctgcg tatggaaggc
      421 gctgtaaatg gacacccgtt cgcgattgaa ggagttggcc ttgggaagcc tttcgaggga
      481 aaacagagta tagaccttaa agtcaaagaa ggcggacctc tgcctttcgc ctatgacatc
      541 ttgacaactg cgttctgtta cggcaacagg gtattcgcca aatacccaga aaatatagta
      601 gactatttca agcagtcgtt tcctgagggc tactcttggg aacgaagcat gaattacgaa
      661 gacgggggca tttgtaacgc gacaaacgac ataaccctgg atggtgactg ttatatctat
      721 gaaattcgat ttgatggtgt gaactttcct gccaatggtc cagttatgca gaagaggact
      781 gtgaaatggg agccatccac tgagaaattg tatgtgcgtg atggagtgct gaagggtgat
      841 gttaacatgg ctctgtcgct tgaaggaggt ggccattacc gatgtgacgt taaaactact
      901 tataaagcta agaagtaaga attctgcaga tatccatcac actggcggcc gctcgagcat
      961 gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc
     1021 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1081 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1141 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1201 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1261 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1321 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1381 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1441 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1501 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1561 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1621 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1681 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     1741 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1801 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     1861 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     1921 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     1981 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     2041 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     2101 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2161 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2221 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2281 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2341 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2401 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2461 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2521 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2581 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2641 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2701 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2761 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2821 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2881 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     2941 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3001 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3061 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     3121 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3181 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     3241 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3301 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3361 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3421 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3481 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3541 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3601 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3661 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3721 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3781 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3841 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3901 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3961 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4021 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4081 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4141 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4201 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4261 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4321 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4381 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4441 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4501 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4561 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4621 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4681 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4741 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4801 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4861 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4921 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4981 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5041 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5101 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5161 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5221 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5281 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5341 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5401 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
     5461 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
     5521 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
     5581 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
     5641 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
     5701 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     5761 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     5821 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     5881 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     5941 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     6001 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     6061 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     6121 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     6181 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     6241 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca
     6301 ctatagggag acccaagctt gccgccacc
//
