LOCUS       sequence-402597-        7254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of nanocage I3-01 Halo-FRB-fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7254)
  AUTHORS   Fung HKH, Hayashi Y, Salo VT, Babenko A, Zagoriy I, Brunner A, 
            Ellenberg J, Muller CW, Cuylen-Haering S, Mahamid J
  TITLE     Genetically encoded multimeric tags for subcellular protein 
            localization in cryo-EM.
  JOURNAL   Nat Methods. 2023 Dec;20(12):1900-1908. doi: 
            10.1038/s41592-023-02053-0. Epub 2023 Nov 6.
  PUBMED    37932397
REFERENCE   2  (bases 1 to 7254)
  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: 
            "10.1038/s41592-023-02053-0"; journalName: "Nat Methods"; date: 
            "2023-12"; volume: "20"; issue: "12"; pages: "1900-1908"
COMMENT     Sequence Label: pcDNA3.1_FRB-GEM23-Halo
FEATURES             Location/Qualifiers
     source          1..7254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(7..24)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(178..766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(258..277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(937..1728,1729..1797))
                     /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     1560..1579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1798..1902)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1977..1996)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2168..2547
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2548..2751
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2702..2722
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2796..2815
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2796..2814
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2849..3127
                     /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             3827..4714
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     primer_bind     complement(4778..4795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4784..5008
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5054..5482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5141..5160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5351..5372
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5491..5511)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5496..5825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5676..5811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5738..5757
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5892..6686
                     /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(5946..5965)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6556..6575
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6860..6981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6897..6916)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6951..6970
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7030..7046)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7030..7046)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7043..7065)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7054..7070
                     /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(7078..7084,7085..7102,7103..7108))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7123..7144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
       61 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      121 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      181 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      241 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      301 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      361 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      421 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      481 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      541 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      601 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      661 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      721 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
      781 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
      841 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
      901 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
      961 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1021 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1081 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1141 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1201 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1261 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1321 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     1381 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     1441 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     1501 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     1561 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     1621 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     1681 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     1741 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     1801 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     1861 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     1921 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     1981 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     2041 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     2101 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     2161 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     2221 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     2281 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     2341 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     2401 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     2461 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     2521 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     2581 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     2641 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     2701 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta
     2761 actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc
     2821 caagctggct agcgccacca tgaagcttat cctctggcat gagatgtggc atgaaggcct
     2881 ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct
     2941 ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa catcctttaa
     3001 tcaggcctat ggtcgagatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc
     3061 agggaatgtc aaggacctcc tccaagcctg ggacctctat tatcatgtgt tccgacgaat
     3121 ctcaaagggc ggcggaggca gcggcggcgg atctggcggc ggcagcggat ccaaaatgga
     3181 agagctgttt aagaagcaca agatcgtggc cgtgctgcgg gccaacagcg tggaagaggc
     3241 caagaagaaa gctctcgccg tctttctggg aggcgtgcac ctgatcgaga ttacattcac
     3301 cgtgcccgac gccgacaccg tgatcaagga actgagcttc ctgaaagaga tgggcgccat
     3361 catcggcgct ggcaccgtga ccagcgtcga gcagtgcaga aaggccgtgg agagcggcgc
     3421 cgagttcatc gtgtcccctc atctggacga ggaaatcagc cagttctgca aggaaaaggg
     3481 cgtgttctac atgcctggcg tgatgacccc taccgagctg gtgaaggcca tgaaactggg
     3541 ccacaccatc ctgaagctgt tccccggaga ggtggtgggc ccccagtttg tgaaggccat
     3601 gaagggccct ttcccaaacg tgaaattcgt gcctacaggc ggcgtgaacc tggataatgt
     3661 ttgtgaatgg ttcaaggccg gcgttctggc tgtgggagtg ggctctgccc tggtcaaggg
     3721 aacacctgtg gaagtggccg agaaggctaa ggccttcgtg gaaaagatca gaggctgcac
     3781 agagggtacc ggaagcgccg gctctgccgc tggcagcggc gagggcgcag aaatcggtac
     3841 tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga
     3901 tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta
     3961 cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct
     4021 gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg
     4081 cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga
     4141 ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat
     4201 tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg
     4261 cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa
     4321 cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat
     4381 ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc
     4441 aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat
     4501 ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct
     4561 gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga
     4621 catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat
     4681 cgcgcgctgg ctgtctactc tggagatttc cggttaggat atccagcaca gtggcggccg
     4741 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     4801 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4861 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4921 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4981 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     5041 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5101 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5401 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5461 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     5521 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5581 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5641 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5701 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5761 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5821 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     5881 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5941 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     6001 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     6061 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     6121 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     6181 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     6241 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     6301 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     6361 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     6421 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     6481 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     6541 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     6601 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     6661 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     6721 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     6781 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     6841 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     6901 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     6961 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     7021 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     7081 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     7141 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     7201 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttc
//
