LOCUS       sequence-254202-        7209 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 7209)
  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 7209)
  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: JB4
FEATURES             Location/Qualifiers
     source          1..7209
                     /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(1558..1911,1912..1914,1915..2523)
                     /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             2554..2580
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2800..2817)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2806..3030
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3076..3504
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3163..3182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3373..3394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3513..3533)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3518..3847
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3698..3833
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3760..3779
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3914..4708
                     /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(3968..3987)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4578..4597
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4882..5003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4919..4938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4973..4992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5052..5068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5052..5068)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5065..5087)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5076..5092
                     /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(5100..5106,5107..5124,5125..5130))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5145..5166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5283..5300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5454..6042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5534..5553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6213..7004,7005..7073))
                     /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     6836..6855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7074..7178)
                     /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 gtatgtccac gggcggagac ttcgggaatc cgctgaggaa attcaagctg
      961 gtgttcctgg gggagcaaag cgttggaaag aattctttga tcaccagatt catgtatgac
     1021 agttttgaca acacctatca ggcaacaatt ggcattgact ttttatcaaa aactatgtac
     1081 ttggaggatc gaacagtacg attgcaatta tgggacacag caggtcaaga gcggttcagg
     1141 agcttgattc ctagctacat tcgtgactcc actgtggcag ttgttgttta tgatatcaca
     1201 aatgttaact cattccagca aactacaaag tggattgatg atgtcagaac agaaagagga
     1261 agtgatgtta tcatcatgct agtaggaaat aaaacagatc ttgctgacaa gaggcaagtg
     1321 tcaattgagg agggagagag gaaagccaaa gagctgaatg ttatgtttat tgaaactagt
     1381 gcaaaagctg gatacaatgt aaagcagctc tttcgacgtg tagcagcagc tttgccggga
     1441 atggaaagca cacaggacag aagcagagaa gatatgattg acataaaact ggaaaagcct
     1501 caggagcaac cagtcagtga aggaggcccg cgggaattcg gagccggagc tggtaccatg
     1561 gacaaggaca acaccgtgcc cctgaagctg atcgccctgc tggccaacgg cgagttccac
     1621 tctggcgagc agctgggaga gaccctggga atgagcagag ccgccatcaa caagcacatc
     1681 cagacactga gagactgggg agtggacgtg ttcaccgtgc ctggcaaggg ctacagcctg
     1741 cctgagccta tccagctgct gaacgccaag cagatcctgg gacagctgga tggcggaagc
     1801 gtggccgtgc tgcctgtgat cgactccacc aatcagtacc tgctggacag aatcggagag
     1861 ctgaagtccg gcgacgcctg catcgccgag taccagcagg ctggcagagg aggcagagga
     1921 cggaagtggt tcagcccatt cggagccaac ctgtacctgt ccatgttctg gagactggag
     1981 cagggacctg ctgctgccat cggactgagt ctggtgatcg gaatcgtgat ggccgaggtg
     2041 ctgagaaagc tgggagccga caaggtgaga gtgaagtggc ctaatgacct gtacctccag
     2101 gaccgcaagc tggctggcat cctggtggag ctgacaggca agacaggcga tgccgctcag
     2161 atcgtgatcg gagccggaat caacatggcc atgagaagag tggaggagag cgtggtgaac
     2221 cagggctgga tcaccctgca ggaggctggc atcaacctgg accggaacac cctggccgcc
     2281 atgctgatca gagagctgag agccgctctg gagctgttcg agcaggaggg actggctcct
     2341 tacctgagca gatgggagaa gctggacaac ttcatcaaca gacctgtgaa gctgatcatc
     2401 ggcgacaagg aaatcttcgg catctccaga ggaatcgaca agcagggagc tctgctgctg
     2461 gagcaggacg gaatcatcaa gccctggatg ggcggagaaa tctccctgag aagcgcagag
     2521 aagggatccg tcgacggagc cggtgctggc gcatacccct acgacgtgcc cgactacgca
     2581 ggctccgcag ttccagcggt agaaatcacc cacaccttct gggaaaggaa cctgccctct
     2641 gtttctggcc tgctgaagat cattggattt tccacatcag taactgccct ggggtttgtg
     2701 ctgtacaaat acaagctcct gccacggtct tgacccgggg atatccagca cagtggcggc
     2761 cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2821 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2881 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2941 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3001 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3061 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3121 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3181 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3241 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3301 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3361 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3421 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3481 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3661 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3721 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3781 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3841 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3901 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3961 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4021 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4081 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4141 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4201 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4261 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4321 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4381 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4441 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     4501 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     4561 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     4621 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4681 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4741 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4801 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4861 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4921 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4981 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5041 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5101 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5161 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5221 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5641 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5701 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5761 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5821 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5881 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5941 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6001 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6061 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7081 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7201 cctgacgtc
//
