LOCUS       sequence-402603-        7557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Synechococcus elongatus encapsulin Halo-FRB-fusion in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7557)
  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 7557)
  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_GEM2-Halo-FRB
FEATURES             Location/Qualifiers
     source          1..7557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(253..357)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(432..451)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        623..1002
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1003..1206
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1157..1177
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1251..1270
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1251..1269
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1289..1298
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2264..3151
                     /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             3194..3472
                     /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(3536..3553)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3542..3766
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3812..4240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3899..3918)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4109..4130
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4249..4269)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4254..4583
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4434..4569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4496..4515
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4650..5444
                     /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(4704..4723)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5314..5333
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5618..5739
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5655..5674)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5709..5728
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5788..5804)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5788..5804)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5801..5823)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5812..5828
                     /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(5836..5842,5843..5860,5861..5866))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5881..5902
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6019..6036)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6190..6778)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6270..6289)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6949..7557,1..183,184..252))
                     /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 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
       61 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
      121 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
      181 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
      241 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
      301 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
      361 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
      421 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
      481 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      541 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      601 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      661 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      721 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      781 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      841 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      901 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      961 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1021 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1081 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1141 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1201 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
     1261 cactataggg agacccaagc tggctagcgc caccatggga tccaccgata acgcccctca
     1321 gctggccctg cgggatgtgg cagctagaca gctggctaat gccaccaaaa ccgtgcccca
     1381 actgcgcacc atcacaccca gatggctggt cagactgctg cactggaccc ctgtggaagc
     1441 cggcatctac cgggttaacc aggtgaagga cgcctcccag attaccgtcg cttgtagcga
     1501 gagagatgag agcgagctgc ccgaaacctt cgtggactac atcgacaatc ctagagagta
     1561 cctgctgagc gccgtgaaca cagtggtgga cgtgcatacc agaatcagcg acctgtatag
     1621 caacccccac gaccagatcc gggaacagct cagactgaca atcgagatca tgaaagaaag
     1681 acaagagtcc gagctgatca acagcagaga atacggcctg ctgaacaacg tggcccccgg
     1741 ccagctggtg cacacaagaa acggcgcccc aacacctgac gacctggacg agctgctgat
     1801 cagagtgtgg aaggaacctg ctttttttct ggcccaccct caggccatcg ccgccttcgg
     1861 cagagagtgc acacggcggg gcgtgccacc tgccacagtg tccctgttcg gcagcagctt
     1921 catcacctgg cggggcgtgc ctctgatccc cagcgacaag gtgcctctgg aaaacggtaa
     1981 gaccaagatc ctgctgctca gggtcggaga gtctagacag ggagttgtgg gactgtacca
     2041 gcctaatctg cctggcgagc agggcatggg cctgtctgtg cggttcatgg gcatcaaccg
     2101 gaaggccctg gccagctacc tggtgtctct gtactgcagc ctggccgtgc tgaccgatga
     2161 tgccctggcc gtgctggata acgtggacgt gacccagtac cacacctaca gatacaacgg
     2221 taccggcggc ggaggcagcg gcggcggatc tggcggcggc agcgcagaaa tcggtactgg
     2281 ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt
     2341 tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt
     2401 gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat
     2461 cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt
     2521 catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg
     2581 gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc
     2641 atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga
     2701 gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt
     2761 ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga
     2821 ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa
     2881 cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga
     2941 ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat
     3001 cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat
     3061 cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc
     3121 gcgctggctg tctactctgg agatttccgg taagcttgga agcgccggct ctgccgctgg
     3181 cagcggcgag ggcatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg
     3241 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc
     3301 tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg cctatggtcg
     3361 agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcaagga
     3421 cctcctccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa agtaggatat
     3481 ccagcacagt ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     3541 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3601 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3661 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3721 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     3781 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3841 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3901 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3961 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4021 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4081 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4141 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4201 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     4261 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4321 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4381 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4441 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4501 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     4561 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     4621 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4681 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4741 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4801 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     4861 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4921 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4981 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5041 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5101 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5161 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     5221 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     5281 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     5341 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     5401 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     5461 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     5521 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5581 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     5641 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5701 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     5761 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5821 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5881 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5941 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6001 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     6061 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6121 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6181 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6241 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6301 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6361 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6421 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6481 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6541 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6601 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6661 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6721 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6781 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6841 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6901 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6961 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7021 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7081 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7141 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7201 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7261 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7321 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7381 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7441 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7501 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgccc
//
