LOCUS       sequence-416393-        6639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  targeted actin nucleation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6639)
  AUTHORS   Nakamura H, Rho E, Lee CT, Itoh K, Deng D, Watanabe S, Razavi S, 
            Matsubayashi HT, Zhu C, Jung E, Rangamani P, Watanabe S, Inoue T
  TITLE     ActuAtor, a Listeria-inspired molecular tool for physical 
            manipulation of intracellular organizations through de novo actin 
            polymerization.
  JOURNAL   Cell Rep. 2023 Sep 2:113089. doi: 10.1016/j.celrep.2023.113089.
  PUBMED    37734382
REFERENCE   2  (bases 1 to 6639)
  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: "Cell Rep. 
            2023 Sep 2:113089. doi: 10.1016/j.celrep.2023.113089."
COMMENT     Sequence Label: EYFP-FKBP-N-WASP
FEATURES             Location/Qualifiers
     source          1..6639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..114
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      156..165
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(204..407)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(233..253)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        408..711
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(886..1474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(966..985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1803..1850
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1875..1894
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2082..2876)
                     /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             complement(2082..2876)
                     /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(2193..2212)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2803..2822
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2911..3268)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2925..3060
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3077..3268
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3225..3245
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3270..3374)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3401..3856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3488..3507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3698..3719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3863..3984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3874..3893)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3928..3947
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5749..6069)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(join(6082..6639,1..153,154..156,157..159))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6124..6145)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6473..6492
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
       61 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
      121 caccaccccg gtgaacagct cctcgccctt gctcaccatg gtggcgaccg gtagcgctag
      181 cggatctgac ggttcactaa accagctctg cttatataga cctcccaccg tacacgccta
      241 ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt
      301 tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag
      361 tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg
      421 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
      481 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
      541 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
      601 tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg
      661 gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc
      721 atatatgggc tatgaactaa tgaccccgta attgattact attaataact aatgcatggc
      781 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
      841 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
      901 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
      961 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     1021 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     1081 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     1141 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     1201 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     1261 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     1321 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     1381 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     1441 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     1501 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     1561 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     1621 atatgagtaa cctgaggcta tggcagggcc tgccgccccg acgttggctg cgagccctgg
     1681 gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg cgtattggcc
     1741 ccaatggggt ctcggtgggg tatcgacaga gtgccagccc tgggaccgaa ccccgcgttt
     1801 atgaacaaac gacccaacac cgtgcgtttt attctgtctt tttattgccg tcatagcgcg
     1861 ggttccttcc ggtattgtct ccttccgtgt ttcagttagc ctccccctag ggtgggcgaa
     1921 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     1981 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     2041 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     2101 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     2161 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     2221 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     2281 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     2341 ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     2401 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     2461 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     2521 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     2581 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     2641 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt
     2701 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     2761 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     2821 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     2881 aacgatcctc atcctgtctc ttgatcgatc tttgcaaaag cctaggcctc caaaaaagcc
     2941 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
     3001 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     3061 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     3121 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     3181 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     3241 attccacagc tggttctttc cgcctcagga ctcttccttt ttcaatatta ttgaagcatt
     3301 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3361 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca
     3421 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3481 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3541 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3601 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3661 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3721 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3781 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3841 ttaacgctta caatttacgc cttaagatac attgatgagt ttggacaaac cacaactaga
     3901 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3961 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4021 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     4081 gattatgatc agttatctag atccggtgga tcccgggccc gcggtaccgt cgactgcaga
     4141 attcgaagct tatcagtctt cccattcatc atcatcctca aaatcttcat catcatcttc
     4201 atcctcatct tcgtctgaag aatgaatggc tttgctcctc ttctgcatca cttccattaa
     4261 tgcacctaca attcctgaag tgggtgcagg tgctggtggt gtagactctg gggcatcagt
     4321 tacagatttc agttgaatac cctgtcgtat ctggtctaaa agtgcatccc ttccagagca
     4381 ggacaccggc cgactgttct gttccacttt ttttagctga gcaccctctc taatttgatc
     4441 taaaagagct gctttgcttc ctgcaggagt tggaacttgg tggtcaccat cagaagggag
     4501 gccaggggga ggtggtggcc ctggtggagg tggaggtggc ggcggaggtg gtggtgccac
     4561 tgacccgctc actgacagag gtggaggtgg tggtggaggc cctgaaggtg ctgaggaggg
     4621 aagagctgga agtggaggag ggtacatcct gtttggcggt ggtggagggg ctcctacacc
     4681 tggcctggat ggaggcggtg gtggcggtgc agctgtggga gctcttgaag gtggtggagg
     4741 aggagcccct cttcccctgg caggaggggg aggagggccc gagctatgtg gaggcggggg
     4801 aggagggggc ggccctcccc ttgatggtgg tggaggtggt ggtgcttgcc ttcgcagttc
     4861 atttttaaca gcttcaacac ctcctgtttt ttcaatgaag tcatatataa cttttgatgt
     4921 ttctctgtct ttaagttgtg cctctgagat tccacacata tcaaaaagat tcttcaattc
     4981 tggatccaaa ttattcagat caaagccagt attcggatcc caacccacat gtccaatgtg
     5041 ttggaaattg cttggtgttc caatatctgc cttagttaac ctcttctttt tagcttttcc
     5101 ttttttcttt tctttggtat gggagatgtt gttgatttgc ggaccataaa atctatttgt
     5161 tgtaatttct ggatttttta tgtcaactgt tgccatgggt agattaggac catttggggg
     5221 gtctcgtctt ttctcagatt tcctttgtcg tcgtcccaac aagtctgtaa ctgcttttcg
     5281 gaattttttt gcttcttctt cattggcaaa attaagagca acttgacagg tatctccagc
     5341 aaaggtatga aaatatcctc taggactatt atatacaaag ttattgtata gctcttgttc
     5401 ccacaatagt ttcccatcct tgatatcaaa tattcttaaa aaataagatc tctgtggatt
     5461 gtccttaaca agacaagcaa caccactgca cttctttgac cacatacagt tccgatctgc
     5521 tgcatataac tgtaccactg ccgaagacat ggtcacacat ttcttgccga ggaaggtgaa
     5581 gagggactcg ttctcctgcg gggtgagcag cagggacccc acgttggtga ccctccgcgg
     5641 cggcggcggc tgctgctggc cggagctcat ggaagcttga gctcgaggac caccagcact
     5701 accaccagca ctaccaccag cactaccacc agcactacca ccagcacttt ccagttttag
     5761 aagctccaca tcgaagacga gagtggcatg tggtgggatg atgcctgggt gcccagtggc
     5821 accataggca taatctggag atatagtcag tttggctctc tgacccacac tcatctgggc
     5881 aaccccttct tcccagcctc ggatcacctc ctgcttgcct agcataaact taaagggctt
     5941 gtttctgtcc cgggaggaat caaatttctt tccatcttca agcatcccgg tgtagtgcac
     6001 cacgcaggtc tggccgcgct tggggaaggt gcgcccgtct cctggggaga tggtttccac
     6061 ctgcactcca ccaccagcac tcttgtacag ctcgtccatg ccgagagtga tcccggcggc
     6121 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc
     6181 ggactggtag ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt
     6241 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt
     6301 gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt
     6361 gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     6421 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     6481 gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga
     6541 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact gcaggccgta
     6601 gccgaaggtg gtcacgaggg tgggccaggg cacgggcag
//
