LOCUS       sequence-254236-        7149 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 7149)
  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 7149)
  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: JB57
FEATURES             Location/Qualifiers
     source          1..7149
                     /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(1498..1851,1852..1854,1855..2463)
                     /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             2494..2520
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2740..2757)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2746..2970
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3016..3444
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3103..3122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3313..3334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3453..3473)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3458..3787
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3638..3773
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3700..3719
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3854..4648
                     /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(3908..3927)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4518..4537
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4822..4943
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4859..4878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4913..4932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4992..5008)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4992..5008)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5005..5027)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5016..5032
                     /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(5040..5046,5047..5064,5065..5070))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5085..5106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5223..5240)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5394..5982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5474..5493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6153..6944,6945..7013))
                     /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     6776..6795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7014..7118)
                     /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 gtatgcagac aattaagtgt gttgttgtgg gcgatggtgc tgttggtaaa
      961 aactgtctcc tgatatccta cacaacaaac aaatttccat cggaatatgt accgactgtt
     1021 tttgacaact atgcagtcac agttatgatt ggtggagaac catatactct tggacttttt
     1081 gatactgcag ggcaagagga ttatgacaga ttacgaccgc tgagttatcc acaaacagat
     1141 gtatttctag tctgtttttc agtggtctct ccatcttcat ttgaaaacgt gaaagaaaag
     1201 tgggtgcctg agataactca ccactgtcca aagactcctt tcttgcttgt tgggactcaa
     1261 attgatctca gagatgaccc ctctactatt gagaaacttg ccaagaacaa acagaagcct
     1321 atcactccag agactgctga aaagctggcc cgtgacctga aggctgtcaa gtatgtggag
     1381 tgttctgcac ttacacagag aggtctgaag aatgtgtttg atgaggctat cctagctgcc
     1441 ctcgagcctc cggaaactca acccaaaagg aaggaattcg gagccggagc tggtaccatg
     1501 gacaaggaca acaccgtgcc cctgaagctg atcgccctgc tggccaacgg cgagttccac
     1561 tctggcgagc agctgggaga gaccctggga atgagcagag ccgccatcaa caagcacatc
     1621 cagacactga gagactgggg agtggacgtg ttcaccgtgc ctggcaaggg ctacagcctg
     1681 cctgagccta tccagctgct gaacgccaag cagatcctgg gacagctgga tggcggaagc
     1741 gtggccgtgc tgcctgtgat cgactccacc aatcagtacc tgctggacag aatcggagag
     1801 ctgaagtccg gcgacgcctg catcgccgag taccagcagg ctggcagagg aggcagagga
     1861 cggaagtggt tcagcccatt cggagccaac ctgtacctgt ccatgttctg gagactggag
     1921 cagggacctg ctgctgccat cggactgagt ctggtgatcg gaatcgtgat ggccgaggtg
     1981 ctgagaaagc tgggagccga caaggtgaga gtgaagtggc ctaatgacct gtacctccag
     2041 gaccgcaagc tggctggcat cctggtggag ctgacaggca agacaggcga tgccgctcag
     2101 atcgtgatcg gagccggaat caacatggcc atgagaagag tggaggagag cgtggtgaac
     2161 cagggctgga tcaccctgca ggaggctggc atcaacctgg accggaacac cctggccgcc
     2221 atgctgatca gagagctgag agccgctctg gagctgttcg agcaggaggg actggctcct
     2281 tacctgagca gatgggagaa gctggacaac ttcatcaaca gacctgtgaa gctgatcatc
     2341 ggcgacaagg aaatcttcgg catctccaga ggaatcgaca agcagggagc tctgctgctg
     2401 gagcaggacg gaatcatcaa gccctggatg ggcggagaaa tctccctgag aagcgcagag
     2461 aagggatccg tcgacggagc cggtgctggc gcatacccct acgacgtgcc cgactacgca
     2521 ggctccgcag ttccagcggt agaaatcacc cacaccttct gggaaaggaa cctgccctct
     2581 gtttctggcc tgctgaagat cattggattt tccacatcag taactgccct ggggtttgtg
     2641 ctgtacaaat acaagctcct gccacggtct tgacccgggg atatccagca cagtggcggc
     2701 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2761 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2821 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2881 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2941 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3001 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3061 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3121 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3181 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3241 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3301 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3361 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3421 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3481 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3541 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3601 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3661 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3721 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3781 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3841 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3901 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3961 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4021 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4081 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4141 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4201 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4261 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4321 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4381 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4441 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4501 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4561 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4621 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4681 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4741 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4801 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4861 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4921 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4981 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5041 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5101 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5161 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5221 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5281 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5341 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5401 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5461 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5521 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5581 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5641 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5701 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5761 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5821 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5881 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5941 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6001 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6061 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6121 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6181 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6241 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6301 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6361 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6421 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6481 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6541 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6601 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6661 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6721 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6781 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6841 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6901 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6961 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7021 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7081 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7141 cctgacgtc
//
