LOCUS       sequence-254203-        7155 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 7155)
  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 7155)
  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: JB68
FEATURES             Location/Qualifiers
     source          1..7155
                     /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(1504..1857,1858..1860,1861..2469)
                     /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             2500..2526
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2746..2763)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2752..2976
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3022..3450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3109..3128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3319..3340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3459..3479)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3464..3793
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3644..3779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3706..3725
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3860..4654
                     /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(3914..3933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4524..4543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4828..4949
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4865..4884)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4919..4938
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4998..5014)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4998..5014)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5011..5033)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5022..5038
                     /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(5046..5052,5053..5070,5071..5076))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5091..5112
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5229..5246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5400..5988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5480..5499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6159..6950,6951..7019))
                     /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     6782..6801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7020..7124)
                     /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 gtatggctgc catccggaag aaactggtga ttgttggtga tggagcctgt
      961 ggaaagacat gcttgctcat agtcttcagc aaggaccagt tcccagaggt gtatgtgccc
     1021 acagtgtttg agaactatgt ggcagatatc gaggtggatg gaaagcaggt agagttggct
     1081 ttgtgggaca cagctgggct ggaagattat gatcgcctga ggcccctctc ctacccagat
     1141 accgatgtta tactgatgtg tttttccatc gacagccctg atagtttaga aaacatccca
     1201 gaaaagtgga ccccagaagt caagcatttc tgtcccaacg tgcccatcat cctggttggg
     1261 aataagaagg atcttcggaa tgatgagcac acaaggcggg agctagccaa gatgaagcag
     1321 gagccggtga aacctgaaga aggcagagat atggcaaaca ggattggcgc ttttgggtac
     1381 atggagtgtt cagcaaagac caaagatgga gtgagagagg tttttgaaat ggctacgaga
     1441 gctgctctgc aagctagacg tgggaagaaa aaatctggtg aattcggagc cggagctggt
     1501 accatggaca aggacaacac cgtgcccctg aagctgatcg ccctgctggc caacggcgag
     1561 ttccactctg gcgagcagct gggagagacc ctgggaatga gcagagccgc catcaacaag
     1621 cacatccaga cactgagaga ctggggagtg gacgtgttca ccgtgcctgg caagggctac
     1681 agcctgcctg agcctatcca gctgctgaac gccaagcaga tcctgggaca gctggatggc
     1741 ggaagcgtgg ccgtgctgcc tgtgatcgac tccaccaatc agtacctgct ggacagaatc
     1801 ggagagctga agtccggcga cgcctgcatc gccgagtacc agcaggctgg cagaggaggc
     1861 agaggacgga agtggttcag cccattcgga gccaacctgt acctgtccat gttctggaga
     1921 ctggagcagg gacctgctgc tgccatcgga ctgagtctgg tgatcggaat cgtgatggcc
     1981 gaggtgctga gaaagctggg agccgacaag gtgagagtga agtggcctaa tgacctgtac
     2041 ctccaggacc gcaagctggc tggcatcctg gtggagctga caggcaagac aggcgatgcc
     2101 gctcagatcg tgatcggagc cggaatcaac atggccatga gaagagtgga ggagagcgtg
     2161 gtgaaccagg gctggatcac cctgcaggag gctggcatca acctggaccg gaacaccctg
     2221 gccgccatgc tgatcagaga gctgagagcc gctctggagc tgttcgagca ggagggactg
     2281 gctccttacc tgagcagatg ggagaagctg gacaacttca tcaacagacc tgtgaagctg
     2341 atcatcggcg acaaggaaat cttcggcatc tccagaggaa tcgacaagca gggagctctg
     2401 ctgctggagc aggacggaat catcaagccc tggatgggcg gagaaatctc cctgagaagc
     2461 gcagagaagg gatccgtcga cggagccggt gctggcgcat acccctacga cgtgcccgac
     2521 tacgcaggct ccgcagttcc agcggtagaa atcacccaca ccttctggga aaggaacctg
     2581 ccctctgttt ctggcctgct gaagatcatt ggattttcca catcagtaac tgccctgggg
     2641 tttgtgctgt acaaatacaa gctcctgcca cggtcttgac ccggggatat ccagcacagt
     2701 ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2761 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2821 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2881 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2941 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3001 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3061 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3121 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3181 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3241 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3301 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3361 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3421 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3661 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3721 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3781 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3841 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3901 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3961 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4021 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4081 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4141 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4201 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4261 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4321 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4381 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4441 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4501 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4561 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4621 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4681 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4741 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4801 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4861 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4921 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4981 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     5041 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5101 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5161 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5461 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5581 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5821 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7021 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7081 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7141 gtgccacctg acgtc
//
