LOCUS       sequence-254217-        7272 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 7272)
  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 7272)
  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: JB25
FEATURES             Location/Qualifiers
     source          1..7272
                     /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(1621..1974,1975..1977,1978..2586)
                     /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             2617..2643
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2863..2880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2869..3093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3139..3567
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3226..3245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3436..3457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3576..3596)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3581..3910
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3761..3896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3823..3842
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3977..4771
                     /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(4031..4050)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4641..4660
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4945..5066
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4982..5001)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5036..5055
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5115..5131)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5115..5131)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5128..5150)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5139..5155
                     /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(5163..5169,5170..5187,5188..5193))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5208..5229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5346..5363)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5517..6105)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5597..5616)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6276..7067,7068..7136))
                     /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     6899..6918
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7137..7241)
                     /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 gtgggggaat ggctgaggag atggagtcgt cgctcgaggc aagcttttcg
      961 tccagcgggg cagtgtcagg ggcctcaggg tttttgcctc ctgcccgctc ccgcatcttc
     1021 aagataatcg tgatcggcga ctccaatgtg ggcaagaact gcctgaccta ccgcttctgc
     1081 gctggccgct tccccgaccg caccgaggcc acgatagggg tggatttccg agaacgagcg
     1141 gtggagattg atggggagcg catcaagatc cagctatggg acacagcagg acaagaacga
     1201 ttcagaaaga gcatggttca gcactactac agaaatgtac atgctgttgt cttcgtgtat
     1261 gatatgacca acatggctag ttttcatagc ctaccatctt ggatagaaga atgcaaacaa
     1321 catttgctag ccaatgatat accacggatt cttgttggaa ataaatgtga cttgagaagt
     1381 gccatacagg tacccacaga cttggcacaa aaatttgctg acacacacag tatgcctttg
     1441 tttgaaacgt ctgctaaaaa ccccaatgat aatgaccatg tggaagctat atttatgacc
     1501 ttggctcata agcttaagag ccacaaacca ttaatgctta gtcagccccc tgataatgga
     1561 attatcctga agcctgaacc aaagcctgca atgacggaat tcggagccgg agctggtacc
     1621 atggacaagg acaacaccgt gcccctgaag ctgatcgccc tgctggccaa cggcgagttc
     1681 cactctggcg agcagctggg agagaccctg ggaatgagca gagccgccat caacaagcac
     1741 atccagacac tgagagactg gggagtggac gtgttcaccg tgcctggcaa gggctacagc
     1801 ctgcctgagc ctatccagct gctgaacgcc aagcagatcc tgggacagct ggatggcgga
     1861 agcgtggccg tgctgcctgt gatcgactcc accaatcagt acctgctgga cagaatcgga
     1921 gagctgaagt ccggcgacgc ctgcatcgcc gagtaccagc aggctggcag aggaggcaga
     1981 ggacggaagt ggttcagccc attcggagcc aacctgtacc tgtccatgtt ctggagactg
     2041 gagcagggac ctgctgctgc catcggactg agtctggtga tcggaatcgt gatggccgag
     2101 gtgctgagaa agctgggagc cgacaaggtg agagtgaagt ggcctaatga cctgtacctc
     2161 caggaccgca agctggctgg catcctggtg gagctgacag gcaagacagg cgatgccgct
     2221 cagatcgtga tcggagccgg aatcaacatg gccatgagaa gagtggagga gagcgtggtg
     2281 aaccagggct ggatcaccct gcaggaggct ggcatcaacc tggaccggaa caccctggcc
     2341 gccatgctga tcagagagct gagagccgct ctggagctgt tcgagcagga gggactggct
     2401 ccttacctga gcagatggga gaagctggac aacttcatca acagacctgt gaagctgatc
     2461 atcggcgaca aggaaatctt cggcatctcc agaggaatcg acaagcaggg agctctgctg
     2521 ctggagcagg acggaatcat caagccctgg atgggcggag aaatctccct gagaagcgca
     2581 gagaagggat ccgtcgacgg agccggtgct ggcgcatacc cctacgacgt gcccgactac
     2641 gcaggctccg cagttccagc ggtagaaatc acccacacct tctgggaaag gaacctgccc
     2701 tctgtttctg gcctgctgaa gatcattgga ttttccacat cagtaactgc cctggggttt
     2761 gtgctgtaca aatacaagct cctgccacgg tcttgacccg gggatatcca gcacagtggc
     2821 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2881 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2941 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3001 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3061 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3121 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3181 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3241 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3301 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3361 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3421 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3481 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3541 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3601 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3661 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3721 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3781 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3841 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3901 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3961 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4021 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4081 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4141 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4201 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4261 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4321 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4381 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4441 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4501 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4561 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4621 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4681 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4741 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4801 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4861 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4921 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4981 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5041 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5101 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5161 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5221 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5281 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5341 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5401 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5461 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5521 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5581 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5641 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5701 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5761 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5821 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5881 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5941 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6001 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6061 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6121 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6181 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6241 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6301 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6361 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6421 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6481 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6541 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6601 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6661 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6721 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6781 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6841 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6901 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6961 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7021 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7081 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7141 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7201 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7261 ccacctgacg tc
//
