LOCUS       sequence-402599-        7434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Mycobacterium hassiacum encapsulin Halo-FRB-fusion in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7434)
  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 7434)
  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_GEM22-Halo-FRB
FEATURES             Location/Qualifiers
     source          1..7434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        58..437
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        438..641
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     592..612
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     686..705
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        686..704
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      724..733
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1576..2463
                     /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             2506..2784
                     /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(2848..2865)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2854..3078
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3124..3552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3211..3230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3421..3442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3561..3581)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3566..3895
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3746..3881
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3808..3827
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3962..4756
                     /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(4016..4035)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4626..4645
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5021..5040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5100..5116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5100..5116)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5113..5135)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5124..5140
                     /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(5148..5154,5155..5172,5173..5178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5193..5214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5331..5348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5502..6090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5582..5601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6261..7052,7053..7121))
                     /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     6884..6903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7122..7226)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7301..7320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
       61 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      121 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      181 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      421 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      481 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      541 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      601 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
      661 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
      721 agcgccacca tgggatccaa caatctgtac cgggagctgg ccccagtgac cgacgccgcc
      781 tggcacgaga tcgagactga ggccatcaga acctttaagc ggcacatcgc cggaagaaga
      841 gtggtggatg tttccgagcc atctggcccc gtgacagccg ctgtgtccac cggccacctg
      901 agagacatca gccctcctgg cgacggcgtg gtcgcccacc tgagagagag caagcctctg
      961 gtgcggctgc gggtgccctt caccgtgacc cgcagcgcca ttgacgacgt tgaaagaggc
     1021 agccaggaca gcgactggga ccccgtgaag gccgctgcta aaaagctggc ctttgtggaa
     1081 gatagagcca tcttcgaggg ctatcctgct gcccagatcg acggcatccg gcaatgtacc
     1141 agcaaccctg tgctgcagct gcctgatgat gccagagaca tcaccgatgt gatcgcccag
     1201 gccctgagcg agctgcggct tgccggcgtc gacggccctt acagcgtgct gctgagcgcc
     1261 gaggtgtaca ccaaggtgtc tgaaacaacc gagcacggct accccatcag ggaacatctg
     1321 aacagactcg tggacggaga catcatctgg gcccctgcca tcgacggcgc cttcgtgctg
     1381 tctaccagag gcggagattt cgacctgaga ctgggaacag acgtcagcat cggctacctg
     1441 agccacgacg ctgaaaccgt gcagctgtac ctggaagaga cactgacatt cctgtgctac
     1501 acatctgaag cctccgtggc tctgacccct ggtaccggcg gcggaggcag cggcggcgga
     1561 tctggcggcg gcagcgcaga aatcggtact ggctttccat tcgaccccca ttatgtggaa
     1621 gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc gcgatggcac ccctgtgctg
     1681 ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca acatcatccc gcatgttgca
     1741 ccgacccatc gctgcattgc tccagacctg atcggtatgg gcaaatccga caaaccagac
     1801 ctgggttatt tcttcgacga ccacgtccgc ttcatggatg ccttcatcga agccctgggt
     1861 ctggaagagg tcgtcctggt cattcacgac tggggctccg ctctgggttt ccactgggcc
     1921 aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg agttcatccg ccctatcccg
     1981 acctgggacg aatggccaga atttgcccgc gagaccttcc aggccttccg caccaccgac
     2041 gtcggccgca agctgatcat cgatcagaac gtttttatcg agggtacgct gccgatgggt
     2101 gtcgtccgcc cgctgactga agtcgagatg gaccattacc gcgagccgtt cctgaatcct
     2161 gttgaccgcg agccactgtg gcgcttccca aacgagctgc caatcgccgg tgagccagcg
     2221 aacatcgtcg cgctggtcga agaatacatg gactggctgc accagtcccc tgtcccgaag
     2281 ctgctgttct ggggcacccc aggcgttctg atcccaccgg ccgaagccgc tcgcctggcc
     2341 aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg gtctgaatct gctgcaagaa
     2401 gacaacccgg acctgatcgg cagcgagatc gcgcgctggc tgtctactct ggagatttcc
     2461 ggtaagcttg gaagcgccgg ctctgccgct ggcagcggcg agggcatcct ctggcatgag
     2521 atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa
     2581 ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg
     2641 aaggaaacat cctttaatca ggcctatggt cgagatttaa tggaggccca agagtggtgc
     2701 aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc aagcctggga cctctattat
     2761 catgtgttcc gacgaatctc aaagtaggat atccagcaca gtggcggccg ctcgagtcta
     2821 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2881 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2941 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3001 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3061 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3121 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3181 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3241 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3301 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3361 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3421 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3481 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3541 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3601 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3661 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3721 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3781 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3841 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3901 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3961 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4021 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4081 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4141 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4201 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4261 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4321 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4381 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4441 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4501 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4561 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4621 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4681 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4741 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4801 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4861 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4921 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5101 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5161 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5221 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5281 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5341 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5401 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5461 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5521 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5581 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5641 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5701 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5761 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5821 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5881 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5941 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6001 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6061 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6121 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6181 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6361 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6601 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6661 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6721 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6781 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6841 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6901 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6961 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7021 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7081 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7141 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7201 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7261 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7321 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7381 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatc
//
