LOCUS       sequence-254252-        7191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression, proximity labelling, mitochondrial 
            relocalisation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7191)
  AUTHORS   Gillingham AK, Bertram J, Begum F, Munro S
  TITLE     In vivo identification of GTPase interactors by mitochondrial 
            relocalization and proximity biotinylation.
  JOURNAL   Elife. 2019 Jul 11;8. pii: 45916. doi: 10.7554/eLife.45916.
  PUBMED    31294692
REFERENCE   2  (bases 1 to 7191)
  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"; journalName: "Elife."; 
            date: "2019-07-11"; volume: "8. pii: 45916. doi"; pages: " 
            10.7554/eLife.45916"
COMMENT     Sequence Label: JB84
FEATURES             Location/Qualifiers
     source          1..7191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1540..1893,1894..1896,1897..2505)
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MDKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLR
                     DWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKS
                     GDACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLR
                     KLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQ
                     GWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLII
                     GDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2536..2562
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2782..2799)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2788..3012
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3058..3486
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3145..3164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3355..3376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3495..3515)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3500..3829
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3680..3815
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3742..3761
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3896..4690
                     /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(3950..3969)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4560..4579
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4864..4985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4901..4920)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4955..4974
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5034..5050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5034..5050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5047..5069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5058..5074
                     /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(5082..5088,5089..5106,5107..5112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5127..5148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5265..5282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5436..6024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5516..5535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6195..6986,6987..7055))
                     /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     6818..6837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7056..7160)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 aagcttaccg gtatggcagg aaaatcatca ctttttaaag taattctcct tggagatggt
      961 ggagttggga agagttcact tatgaacaga tatgtaacta ataagtttga tacccagctc
     1021 ttccatacaa taggtgtgga atttttaaat aaagatttgg aagtggatgg acattttgtt
     1081 accatgcaga tttgggacac ggcaggtctg gagcgattcc gaagcctgag gacaccattt
     1141 tacagaggtt ctgactgctg cctgcttact tttagtgtcg atgattcaca aagcttccag
     1201 aacttaagta actggaagaa agaattcata tattatgcag atgtgaaaga gcctgagagc
     1261 tttccttttg tgattctggg taacaagatt gacataagcg aacggcaggt gtctacagaa
     1321 gaagcccaag cttggtgcag ggacaacggc gactatcctt attttgaaac aagtgcaaaa
     1381 gatgccacaa atgtggcagc agcctttgag gaagcggttc gaagagttct tgctaccgag
     1441 gataggtcag atcatttgat tcagacagac acagtcaatc ttcaccgaaa gcccaagcct
     1501 agctcatctc cgcgggaatt cggagccgga gctggtacca tggacaagga caacaccgtg
     1561 cccctgaagc tgatcgccct gctggccaac ggcgagttcc actctggcga gcagctggga
     1621 gagaccctgg gaatgagcag agccgccatc aacaagcaca tccagacact gagagactgg
     1681 ggagtggacg tgttcaccgt gcctggcaag ggctacagcc tgcctgagcc tatccagctg
     1741 ctgaacgcca agcagatcct gggacagctg gatggcggaa gcgtggccgt gctgcctgtg
     1801 atcgactcca ccaatcagta cctgctggac agaatcggag agctgaagtc cggcgacgcc
     1861 tgcatcgccg agtaccagca ggctggcaga ggaggcagag gacggaagtg gttcagccca
     1921 ttcggagcca acctgtacct gtccatgttc tggagactgg agcagggacc tgctgctgcc
     1981 atcggactga gtctggtgat cggaatcgtg atggccgagg tgctgagaaa gctgggagcc
     2041 gacaaggtga gagtgaagtg gcctaatgac ctgtacctcc aggaccgcaa gctggctggc
     2101 atcctggtgg agctgacagg caagacaggc gatgccgctc agatcgtgat cggagccgga
     2161 atcaacatgg ccatgagaag agtggaggag agcgtggtga accagggctg gatcaccctg
     2221 caggaggctg gcatcaacct ggaccggaac accctggccg ccatgctgat cagagagctg
     2281 agagccgctc tggagctgtt cgagcaggag ggactggctc cttacctgag cagatgggag
     2341 aagctggaca acttcatcaa cagacctgtg aagctgatca tcggcgacaa ggaaatcttc
     2401 ggcatctcca gaggaatcga caagcaggga gctctgctgc tggagcagga cggaatcatc
     2461 aagccctgga tgggcggaga aatctccctg agaagcgcag agaagggatc cgtcgacgga
     2521 gccggtgctg gcgcataccc ctacgacgtg cccgactacg caggctccgc agttccagcg
     2581 gtagaaatca cccacacctt ctgggaaagg aacctgccct ctgtttctgg cctgctgaag
     2641 atcattggat tttccacatc agtaactgcc ctggggtttg tgctgtacaa atacaagctc
     2701 ctgccacggt cttgacccgg ggatatccag cacagtggcg gccgctcgag tctagagggc
     2761 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2821 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2881 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2941 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     3001 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     3061 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3121 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3181 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3241 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3301 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3361 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3421 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3481 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3541 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3601 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3661 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3721 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
     3781 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
     3841 agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat
     3901 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3961 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4021 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4081 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4141 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4201 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4261 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4321 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4381 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4441 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4501 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4561 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4621 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4681 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4741 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4801 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4861 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4921 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4981 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     5041 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5101 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5161 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5221 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5281 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5341 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5401 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5461 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5521 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5581 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5641 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5701 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5761 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5821 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5881 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5941 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6001 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6061 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6121 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6181 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6241 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6301 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6361 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6421 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6481 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6541 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6601 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6661 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6721 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6781 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6841 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6901 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6961 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7021 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7081 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7141 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
