LOCUS       sequence-254245-        7200 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 7200)
  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 7200)
  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: JB97
FEATURES             Location/Qualifiers
     source          1..7200
                     /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(1549..1902,1903..1905,1906..2514)
                     /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             2545..2571
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2791..2808)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2797..3021
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3067..3495
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3154..3173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3364..3385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3504..3524)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3509..3838
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3689..3824
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3751..3770
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3905..4699
                     /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(3959..3978)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4569..4588
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4873..4994
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4910..4929)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4964..4983
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5043..5059)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5043..5059)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5056..5078)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5067..5083
                     /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(5091..5097,5098..5115,5116..5121))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5136..5157
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5274..5291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5445..6033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5525..5544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6204..6995,6996..7064))
                     /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     6827..6846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7065..7169)
                     /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 caacaagagt aagggccaga gctccttggc cctccacaag
      961 gtgatcatgg ttggcagcgt aggcgttggc aagtcagccc tgacgcttca gttcatgtat
     1021 gacgagtttg tagaagacta tgaacctacc aaagctgaca gttatagaaa gaaagtggtt
     1081 cttgatgggg aagaagttca gatagatatt ctggacaccg ctgggcaaga ggactacgca
     1141 gccattcgag ataactactt tcggagtggg gaagggtttc ttcttgtgtt ctcaatcaca
     1201 gaacatgaat cctttacagc aactgccgaa ttcagggaac agattctccg tgtgaaggct
     1261 gaagaagata aaattccact gctcgtcgtg ggaaacaagt ctgacctaga ggagcggagg
     1321 caggtgcctg tggaggaggc caggagtaaa gccgaagagt ggggcgtgca gtacgtggag
     1381 acgtcagcga agacccgggc caacgtggac aaggtgttct ttgacctaat gagagaaatc
     1441 agaacaaaga agatgtcaga aaacaaagac aagaatggca agaaaagcag caagaacaag
     1501 aaaagtttta aagaaagacc gcgggaattc ggagccggag ctggtaccat ggacaaggac
     1561 aacaccgtgc ccctgaagct gatcgccctg ctggccaacg gcgagttcca ctctggcgag
     1621 cagctgggag agaccctggg aatgagcaga gccgccatca acaagcacat ccagacactg
     1681 agagactggg gagtggacgt gttcaccgtg cctggcaagg gctacagcct gcctgagcct
     1741 atccagctgc tgaacgccaa gcagatcctg ggacagctgg atggcggaag cgtggccgtg
     1801 ctgcctgtga tcgactccac caatcagtac ctgctggaca gaatcggaga gctgaagtcc
     1861 ggcgacgcct gcatcgccga gtaccagcag gctggcagag gaggcagagg acggaagtgg
     1921 ttcagcccat tcggagccaa cctgtacctg tccatgttct ggagactgga gcagggacct
     1981 gctgctgcca tcggactgag tctggtgatc ggaatcgtga tggccgaggt gctgagaaag
     2041 ctgggagccg acaaggtgag agtgaagtgg cctaatgacc tgtacctcca ggaccgcaag
     2101 ctggctggca tcctggtgga gctgacaggc aagacaggcg atgccgctca gatcgtgatc
     2161 ggagccggaa tcaacatggc catgagaaga gtggaggaga gcgtggtgaa ccagggctgg
     2221 atcaccctgc aggaggctgg catcaacctg gaccggaaca ccctggccgc catgctgatc
     2281 agagagctga gagccgctct ggagctgttc gagcaggagg gactggctcc ttacctgagc
     2341 agatgggaga agctggacaa cttcatcaac agacctgtga agctgatcat cggcgacaag
     2401 gaaatcttcg gcatctccag aggaatcgac aagcagggag ctctgctgct ggagcaggac
     2461 ggaatcatca agccctggat gggcggagaa atctccctga gaagcgcaga gaagggatcc
     2521 gtcgacggag ccggtgctgg cgcatacccc tacgacgtgc ccgactacgc aggctccgca
     2581 gttccagcgg tagaaatcac ccacaccttc tgggaaagga acctgccctc tgtttctggc
     2641 ctgctgaaga tcattggatt ttccacatca gtaactgccc tggggtttgt gctgtacaaa
     2701 tacaagctcc tgccacggtc ttgacccggg gatatccagc acagtggcgg ccgctcgagt
     2761 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     2821 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2881 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2941 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3001 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     3061 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3121 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3181 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3241 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3301 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3361 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3421 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3481 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3541 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3601 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3661 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3721 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     3781 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3841 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     3901 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3961 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4021 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4081 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4141 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4201 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4261 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4321 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4381 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4441 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4501 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4561 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4621 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4681 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4741 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4801 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4861 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4921 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4981 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     5041 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5101 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5161 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5221 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5281 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5341 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5401 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5461 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5521 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5581 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5641 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5701 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5761 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5821 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5881 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5941 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6001 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6061 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6121 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6181 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6241 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6301 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6361 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6421 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6481 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6541 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6601 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6661 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6721 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6781 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6841 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6901 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6961 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7021 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7081 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7141 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
