LOCUS       sequence-402600-        7479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Rhodospirillum rubrum encapsulin Halo-FRB-fusion in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7479)
  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 7479)
  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_GEM7-Halo-FRB
FEATURES             Location/Qualifiers
     source          1..7479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(110..214)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(289..308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        480..859
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        860..1063
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1014..1034
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1108..1127
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1108..1126
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1146..1155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2043..2930
                     /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             2973..3251
                     /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(3315..3332)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3321..3545
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3591..4019
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3678..3697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3888..3909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4028..4048)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4033..4362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4213..4348
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4275..4294
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4429..5223
                     /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(4483..4502)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5093..5112
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5397..5518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5434..5453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5488..5507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5567..5583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5567..5583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5580..5602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5591..5607
                     /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(5615..5621,5622..5639,5640..5645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5660..5681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5798..5815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5969..6557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6049..6068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6728..7479,1..40,41..109))
                     /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     7351..7370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
       61 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      121 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      181 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
      241 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      301 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      361 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      421 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      481 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      541 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      601 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      661 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      721 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      781 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      841 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      901 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      961 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1021 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     1081 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     1141 ctagcgccac catgggatcc aacgacctga tgagagacct ggctccaatt agcgccaagg
     1201 cctgggccga aatcgagaca gaggcccggg gcaccctgac cgtgaccctg gccgccagaa
     1261 aagtggtgga cttcaagggc cccctgggct gggacgcctc ctctgtgtcc ctgggcagaa
     1321 ccgaggccct ggctgaggaa cctaaggccg ccggcagcgc cgctgtggtc acagtgcgga
     1381 agcgggctgt gcagcctctg atcgagctgt gtgtgccatt caccctgaaa agagctgaac
     1441 tggaagctat cgcgagagga gcctctgatg ccgatctgga ccctgttatc gaggccgcta
     1501 gagccatcgc catcgccgag gaccgggccg tctttcatgg cttcgccgca ggcggcatca
     1561 caggcatcgg agaagccagc gctgaacacg ccctggacct gcctgccgac ctcgccgact
     1621 tccccggcgt gctggtgcgc gccctcgctg tgctgcggga cagaggcgtg gacggcccct
     1681 acgccctggt cctgggaagg accgtgtacc agcagctgat ggaaaccaca acccctggcg
     1741 gctaccctgt gcttcaacac gtgcggagac tgttcgaggg ccctctgatc tgggcccctg
     1801 gagttgatgg cgccatgctg atcagccaga gaggcggcga ctttgagctg acagtgggca
     1861 gagatttcag catcggatac cacgaccacg atgcccagag cgtgcacctg tacctgcagg
     1921 agagcatgac cttcagatgc ctgggccctg aggccgccgt gcccctgaga ggcctgagcc
     1981 aggccgccac caaggccggt accggcggcg gaggcagcgg cggcggatct ggcggcggca
     2041 gcgcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc
     2101 gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta
     2161 acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct
     2221 gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct
     2281 tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg
     2341 tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag
     2401 agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat
     2461 ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc
     2521 tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc
     2581 tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc
     2641 cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc
     2701 tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg
     2761 gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta
     2821 actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc
     2881 tgatcggcag cgagatcgcg cgctggctgt ctactctgga gatttccggt aagcttggaa
     2941 gcgccggctc tgccgctggc agcggcgagg gcatcctctg gcatgagatg tggcatgaag
     3001 gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc atgtttgagg
     3061 tgctggagcc cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct
     3121 ttaatcaggc ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga
     3181 aatcagggaa tgtcaaggac ctcctccaag cctgggacct ctattatcat gtgttccgac
     3241 gaatctcaaa gtaggatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta
     3301 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3361 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3421 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3481 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3541 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3601 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3661 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3721 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3781 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3841 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3901 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3961 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     4021 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4081 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4141 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4201 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4261 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4321 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4381 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4441 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4501 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4561 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4621 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4681 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4741 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4801 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4861 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4921 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4981 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5041 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5101 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5161 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5221 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5281 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5341 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5401 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5461 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5521 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5581 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5641 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5701 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5761 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5821 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5881 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5941 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6001 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6061 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6121 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6181 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6241 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6301 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6361 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6421 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6481 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6541 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6601 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6661 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6721 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6781 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6841 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6901 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6961 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7021 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7081 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     7141 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     7201 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7261 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7321 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7381 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7441 tgagatccag ttcgatgtaa cccactcgtg cacccaact
//
