LOCUS       sequence-254183-        7134 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 7134)
  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 7134)
  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: JB88
FEATURES             Location/Qualifiers
     source          1..7134
                     /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(1483..1836,1837..1839,1840..2448)
                     /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             2479..2505
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2725..2742)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2731..2955
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3001..3429
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3088..3107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3298..3319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3438..3458)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3443..3772
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3623..3758
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3685..3704
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3839..4633
                     /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(3893..3912)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4503..4522
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4807..4928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4844..4863)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4898..4917
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4977..4993)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4977..4993)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4990..5012)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5001..5017
                     /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(5025..5031,5032..5049,5050..5055))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5070..5091
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5208..5225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5379..5967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5459..5478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6138..6929,6930..6998))
                     /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     6761..6780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6999..7103)
                     /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 ggatgccgca gtccaagtcc cggaagatcg cgatcctggg ctaccggtct
      961 gtggggaaaa actcattgac gattcaattt gttgaaggcc aatttgtgga ctcctacgat
     1021 ccaaccatag aaaacacttt tacaaagttg atcacagtaa atggacaaga atatcatctt
     1081 caacttgtag acacagccgg gcaagatgaa tattctatct ttcctcagac atactccata
     1141 gatattaatg gctatattct tgtgtattct gttacatcaa tcaaaagttt tgaagtgatt
     1201 aaagttatcc atggcaaatt gttggatatg gtggggaaag tacaaatacc tattatgttg
     1261 gttgggaata agaaagacct gcatatggaa agggtgatca gttatgaaga agggaaagct
     1321 ttggcagaat cttggaatgc agcttttttg gaatcttctg ctaaagaaaa tcagactgct
     1381 gtggatgttt ttcgaaggat aattttggag gcagaaaaaa tggacggggc agcttcacaa
     1441 ggcaagtctt caccgcggga attcggagcc ggagctggta ccatggacaa ggacaacacc
     1501 gtgcccctga agctgatcgc cctgctggcc aacggcgagt tccactctgg cgagcagctg
     1561 ggagagaccc tgggaatgag cagagccgcc atcaacaagc acatccagac actgagagac
     1621 tggggagtgg acgtgttcac cgtgcctggc aagggctaca gcctgcctga gcctatccag
     1681 ctgctgaacg ccaagcagat cctgggacag ctggatggcg gaagcgtggc cgtgctgcct
     1741 gtgatcgact ccaccaatca gtacctgctg gacagaatcg gagagctgaa gtccggcgac
     1801 gcctgcatcg ccgagtacca gcaggctggc agaggaggca gaggacggaa gtggttcagc
     1861 ccattcggag ccaacctgta cctgtccatg ttctggagac tggagcaggg acctgctgct
     1921 gccatcggac tgagtctggt gatcggaatc gtgatggccg aggtgctgag aaagctggga
     1981 gccgacaagg tgagagtgaa gtggcctaat gacctgtacc tccaggaccg caagctggct
     2041 ggcatcctgg tggagctgac aggcaagaca ggcgatgccg ctcagatcgt gatcggagcc
     2101 ggaatcaaca tggccatgag aagagtggag gagagcgtgg tgaaccaggg ctggatcacc
     2161 ctgcaggagg ctggcatcaa cctggaccgg aacaccctgg ccgccatgct gatcagagag
     2221 ctgagagccg ctctggagct gttcgagcag gagggactgg ctccttacct gagcagatgg
     2281 gagaagctgg acaacttcat caacagacct gtgaagctga tcatcggcga caaggaaatc
     2341 ttcggcatct ccagaggaat cgacaagcag ggagctctgc tgctggagca ggacggaatc
     2401 atcaagccct ggatgggcgg agaaatctcc ctgagaagcg cagagaaggg atccgtcgac
     2461 ggagccggtg ctggcgcata cccctacgac gtgcccgact acgcaggctc cgcagttcca
     2521 gcggtagaaa tcacccacac cttctgggaa aggaacctgc cctctgtttc tggcctgctg
     2581 aagatcattg gattttccac atcagtaact gccctggggt ttgtgctgta caaatacaag
     2641 ctcctgccac ggtcttgacc cggggatatc cagcacagtg gcggccgctc gagtctagag
     2701 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2761 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2821 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2881 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2941 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3001 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3061 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3421 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3481 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3601 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3661 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3721 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3781 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3841 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3901 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3961 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4021 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4081 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4141 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4201 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4261 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4321 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4381 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4441 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4501 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4561 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4621 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4681 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4741 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4801 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4861 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4921 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4981 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5041 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5101 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5221 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5281 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5341 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5401 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5461 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5521 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5581 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5641 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5701 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5761 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5821 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5881 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5941 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6001 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6961 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7021 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7081 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
