LOCUS       sequence-416293-        5406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golgi marker and anchor for FKBP-FRB CID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5406)
  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 5406)
  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: ECFP-FRB-GiantinS
FEATURES             Location/Qualifiers
     source          1..5406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        160..363
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     314..334
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      402..411
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(408..410,411..413,414..1124)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(453..474)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(714..733)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1061..1082
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1137..1415
                     /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    1989..2110
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2026..2045)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2080..2099
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2117..2572)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2254..2275)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2466..2485
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2599..2703
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2705..3062
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2705..2896
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2728..2748)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2913..3048
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3097..3891
                     /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             3097..3891
                     /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(3151..3170)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3761..3780
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4079..4098)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4123..4170
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4499..5087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4988..5007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5262..5406,1..159)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
       61 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      121 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      181 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      241 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      301 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      361 gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg
      421 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      481 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      541 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      601 tgacctgggg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      661 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      721 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
      781 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
      841 actacatcag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
      901 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
      961 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1021 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1081 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggagca agtggaatcc
     1141 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
     1201 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
     1261 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
     1321 aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg
     1381 acctctatta tcatgtgttc cgacgaatct caaagagtgc tggtggtagt gctggtggta
     1441 gtgctggtgg tactggtggt cctcgaggag aaccgcagca aagcttttct gaagctcagc
     1501 agcagctatg caacaccaga caggaagtga atgaattaag gaagctgctg gaagaagaac
     1561 gagaccaaag agtggctgct gagaatgctc tctctgtggc cgaggagcag atcagacggt
     1621 tagagcacag tgaatgggac tcttcccgga ctcctatcat tggctcctgt ggcactcagg
     1681 agcaggcact gttaatagat cttacaagca acagttgtcg aaggacccgg agtggcgttg
     1741 gatggaagcg agtcctgcgt tcactctgtc attcacggac ccgagtgcca cttctagcag
     1801 ccatctactt tctaatgatt catgtcctgc tcattctgtg ttttacgggc catctatagg
     1861 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     1921 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     1981 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     2041 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     2101 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2161 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2221 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2281 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2341 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2401 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2461 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2521 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     2581 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2641 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2701 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2761 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2821 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2881 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2941 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3001 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3061 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3121 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3181 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3241 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     3301 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3361 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3421 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3481 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3541 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3601 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3661 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3721 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3781 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3841 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3901 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3961 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4021 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     4081 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4141 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     4201 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     4261 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     4321 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     4381 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4441 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4501 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4561 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4621 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4681 agaactctgt atcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4741 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4801 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4861 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4921 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4981 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5041 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5101 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5161 tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta atagtaatca
     5221 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     5281 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5341 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5401 taaact
//
