LOCUS       sequence-403329-        7440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Paraburkholderia xenovorans encapsulin Halo-FRB-fusion
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7440)
  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 7440)
  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_GEM4-Halo-FRB
FEATURES             Location/Qualifiers
     source          1..7440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     438..457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(676..780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(855..874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1046..1425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1426..1629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1580..1600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1674..1693
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1674..1692
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1712..1721
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2570..3457
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             3500..3778
                     /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"
     primer_bind     complement(3842..3859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3848..4072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4118..4546
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4205..4224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4415..4436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4555..4575)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4560..4889
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4740..4875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4802..4821
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4956..5750
                     /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(5010..5029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5620..5639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5924..6045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5961..5980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6015..6034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6094..6110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6094..6110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6107..6129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6118..6134
                     /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(6142..6148,6149..6166,6167..6172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6187..6208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6325..6342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6496..7084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6576..6595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7255..7440,1..606,607..675))
                     /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"
ORIGIN
        1 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
       61 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
      121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
      181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      781 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
      841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      901 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      961 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1021 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1081 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1141 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1201 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1261 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1321 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1381 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1441 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1501 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1561 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1621 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     1681 actcactata gggagaccca agctggctag cgccaccatg ggatccaaca acctgcacag
     1741 agagctggct cctatcagca gagccgcatg gtcccagatc gaggacgagg tggccagaac
     1801 cttcagacgc agcgtggccg gtagaagagt ggtcgacgtg aaaggccctg gaggcaccga
     1861 gctttctggc gtgggaacag gccaccagac cgccatcgcc gccccccagc agggcgtggt
     1921 ggccaaactg tccgaggtga agtctctggt ggaactgaca gttcctttcg agctccaaag
     1981 agaggccatc gacagcgtgg tgcggggcgc caaggacgcc gactggcagc ctgccaagga
     2041 agccgctaag caactggcct acgccgaaga tagagctatc ttcgacggct accaggccgc
     2101 cggaatcggc ggcatccggg aaggcagcag caatcctagc ctggccctgc ctgctgatgt
     2161 gtccgactac ccaaacgcca ttagcaacgc cctggaacag ctgcggctgg ccggcgtgga
     2221 cggcccctac agcgtgctgc tgggcgctga tgcttataca gccctgggag aagctagaga
     2281 tcagggctac cccgtgatcg agcacatcaa gcggatcgtg aacggcgaca tcatctgggc
     2341 ccctgccctg gccggcggca gcgtgctgag caccagggga ggcgacttcg agctgcatct
     2401 gggcgaggac ctgagcatcg gatacaccag ccacaccgac acagtggtgc ggctgtacct
     2461 gagagaaacc ctgacctttc tgatgctgac ctctgaggcc tctgtcagcg ttacacaggc
     2521 cgagggtacc ggcggcggag gcagcggcgg cggatctggc ggcggcagcg cagaaatcgg
     2581 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     2641 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     2701 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     2761 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     2821 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     2881 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     2941 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     3001 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     3061 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     3121 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     3181 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     3241 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     3301 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     3361 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     3421 gatcgcgcgc tggctgtcta ctctggagat ttccggtaag cttggaagcg ccggctctgc
     3481 cgctggcagc ggcgagggca tcctctggca tgagatgtgg catgaaggcc tggaagaggc
     3541 atctcgtttg tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc tggagccctt
     3601 gcatgctatg atggaacggg gcccccagac tctgaaggaa acatccttta atcaggccta
     3661 tggtcgagat ttaatggagg cccaagagtg gtgcaggaag tacatgaaat cagggaatgt
     3721 caaggacctc ctccaagcct gggacctcta ttatcatgtg ttccgacgaa tctcaaagta
     3781 ggatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca
     3841 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3901 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3961 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4021 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4081 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     4141 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4201 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4261 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4321 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4381 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4441 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4501 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     4561 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4621 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4681 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4741 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4801 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4861 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4921 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4981 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     5041 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     5101 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     5161 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     5221 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     5281 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     5341 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5401 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5461 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     5521 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     5581 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5641 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5701 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5761 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5821 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5881 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     5941 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     6001 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     6061 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     6121 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6181 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6241 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6301 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6361 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6421 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6661 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6901 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7081 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7141 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7201 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7261 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7321 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7381 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
//
