LOCUS       sequence-254223-        7188 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 7188)
  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 7188)
  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: JB3
FEATURES             Location/Qualifiers
     source          1..7188
                     /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(1537..1890,1891..1893,1894..2502)
                     /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             2533..2559
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2779..2796)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2785..3009
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3055..3483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3142..3161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3352..3373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3492..3512)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3497..3826
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3677..3812
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3739..3758
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3893..4687
                     /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(3947..3966)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4557..4576
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4861..4982
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4898..4917)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4952..4971
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5031..5047)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5031..5047)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5044..5066)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5055..5071
                     /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(5079..5085,5086..5103,5104..5109))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5124..5145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5262..5279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5433..6021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5513..5532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6192..6983,6984..7052))
                     /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     6815..6834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7053..7157)
                     /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 gtatgagtat ggaagattat gatttcctgt tcaaaattgt tttaattggc
      961 aacgctggtg tggggaagaa ttgcctcgtc cgaagattca ctcagggtct tttcccccca
     1021 ggtcaaggag ccacaattgg agttgatttt atgattaaga cagtggagat taatggtgaa
     1081 aaagtaaagc tacagatctg ggacacagca ggtcaagaga gatttcggtc cattacccag
     1141 agttactacc gaagcgccaa tgccttgatc ctcacctatg acattacctg tgaggaatcc
     1201 ttccgttgcc ttcctgagtg gctgcgggag atagaacaat atgccagcaa caaggtcatc
     1261 actgtgttag tgggcaacaa gattgacctg gctgaaagga gagaggtttc ccagcagcga
     1321 gctgaagaat tctcagaagc tcaggacatg tattatctgg agacctcagc caaggaatct
     1381 gataatgtgg agaaactctt ccttgactta gcatgccgac tcatcagtga agccagacag
     1441 aacacacttg tgaacaatgt atcctcaccc ttacctggag aagggaaaag catcagctat
     1501 ttgactccgc gggaattcgg agccggagct ggtaccatgg acaaggacaa caccgtgccc
     1561 ctgaagctga tcgccctgct ggccaacggc gagttccact ctggcgagca gctgggagag
     1621 accctgggaa tgagcagagc cgccatcaac aagcacatcc agacactgag agactgggga
     1681 gtggacgtgt tcaccgtgcc tggcaagggc tacagcctgc ctgagcctat ccagctgctg
     1741 aacgccaagc agatcctggg acagctggat ggcggaagcg tggccgtgct gcctgtgatc
     1801 gactccacca atcagtacct gctggacaga atcggagagc tgaagtccgg cgacgcctgc
     1861 atcgccgagt accagcaggc tggcagagga ggcagaggac ggaagtggtt cagcccattc
     1921 ggagccaacc tgtacctgtc catgttctgg agactggagc agggacctgc tgctgccatc
     1981 ggactgagtc tggtgatcgg aatcgtgatg gccgaggtgc tgagaaagct gggagccgac
     2041 aaggtgagag tgaagtggcc taatgacctg tacctccagg accgcaagct ggctggcatc
     2101 ctggtggagc tgacaggcaa gacaggcgat gccgctcaga tcgtgatcgg agccggaatc
     2161 aacatggcca tgagaagagt ggaggagagc gtggtgaacc agggctggat caccctgcag
     2221 gaggctggca tcaacctgga ccggaacacc ctggccgcca tgctgatcag agagctgaga
     2281 gccgctctgg agctgttcga gcaggaggga ctggctcctt acctgagcag atgggagaag
     2341 ctggacaact tcatcaacag acctgtgaag ctgatcatcg gcgacaagga aatcttcggc
     2401 atctccagag gaatcgacaa gcagggagct ctgctgctgg agcaggacgg aatcatcaag
     2461 ccctggatgg gcggagaaat ctccctgaga agcgcagaga agggatccgt cgacggagcc
     2521 ggtgctggcg cataccccta cgacgtgccc gactacgcag gctccgcagt tccagcggta
     2581 gaaatcaccc acaccttctg ggaaaggaac ctgccctctg tttctggcct gctgaagatc
     2641 attggatttt ccacatcagt aactgccctg gggtttgtgc tgtacaaata caagctcctg
     2701 ccacggtctt gacccgggga tatccagcac agtggcggcc gctcgagtct agagggcccg
     2761 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2821 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2881 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2941 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3001 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3061 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3121 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3181 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3241 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3301 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3361 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3421 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3481 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3541 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3601 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3661 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3721 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     3781 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     3841 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     3901 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     3961 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4021 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4081 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4141 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4201 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4261 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4321 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4381 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4441 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4501 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4561 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4621 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     4681 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     4741 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4801 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4861 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4921 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4981 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5041 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5101 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5161 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5221 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5281 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5341 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5401 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5461 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5521 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5581 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5641 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5701 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5761 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5821 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5881 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5941 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6001 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6061 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6121 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6181 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6241 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6301 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6361 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6421 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6481 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6541 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6601 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6661 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6721 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6781 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6841 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6901 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6961 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7021 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7081 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7141 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
