LOCUS       sequence-254221-        7182 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 7182)
  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 7182)
  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: JB11
FEATURES             Location/Qualifiers
     source          1..7182
                     /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(1531..1884,1885..1887,1888..2496)
                     /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             2527..2553
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2773..2790)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2779..3003
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3049..3477
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3136..3155)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3346..3367
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3486..3506)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3491..3820
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3671..3806
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3733..3752
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3887..4681
                     /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(3941..3960)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4551..4570
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4855..4976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4892..4911)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4946..4965
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5025..5041)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5025..5041)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5038..5060)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5049..5065
                     /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(5073..5079,5080..5097,5098..5103))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5118..5139
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5256..5273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5427..6015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5507..5526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6186..6977,6978..7046))
                     /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     6809..6828
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7047..7151)
                     /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 gtatggacga ggacgtgtta accaccctga agatcctcat catcggcgag
      961 agtggggtgg gcaagtccag cctgctcttg aggttcacag atgatacgtt tgatccagaa
     1021 cttgcagcaa caataggtgt tgactttaag gtgaaaacaa tttcagtgga tggaaataag
     1081 gctaaacttg caatatggga tactgctggt ctagagaggt ttagaacatt aactcccagc
     1141 tattatagag gtgcacaggg tgttatatta gtttatgatg tcacaagaag agatacattt
     1201 gttaaactgg ataattggtt aaatgaattg gaaacatact gtacaagaaa tgacatagta
     1261 aacatgctag ttggaaataa aatcgataag gaaaatcgtg aagtcgatag aaatgaaggc
     1321 ctgaaatttg cacgaaagca ttccatgtta tttatagagg caagtgcaaa aacctgtgat
     1381 ggtgtacaat gtgcctttga agaacttgtt gaaaagatca ttcagacccc tggactgtgg
     1441 gaaagtgaga accagaataa aggagtcaaa ctgtcacaca gggaagaagg ccaaggagga
     1501 ggagccgaat tcggagccgg agctggtacc atggacaagg acaacaccgt gcccctgaag
     1561 ctgatcgccc tgctggccaa cggcgagttc cactctggcg agcagctggg agagaccctg
     1621 ggaatgagca gagccgccat caacaagcac atccagacac tgagagactg gggagtggac
     1681 gtgttcaccg tgcctggcaa gggctacagc ctgcctgagc ctatccagct gctgaacgcc
     1741 aagcagatcc tgggacagct ggatggcgga agcgtggccg tgctgcctgt gatcgactcc
     1801 accaatcagt acctgctgga cagaatcgga gagctgaagt ccggcgacgc ctgcatcgcc
     1861 gagtaccagc aggctggcag aggaggcaga ggacggaagt ggttcagccc attcggagcc
     1921 aacctgtacc tgtccatgtt ctggagactg gagcagggac ctgctgctgc catcggactg
     1981 agtctggtga tcggaatcgt gatggccgag gtgctgagaa agctgggagc cgacaaggtg
     2041 agagtgaagt ggcctaatga cctgtacctc caggaccgca agctggctgg catcctggtg
     2101 gagctgacag gcaagacagg cgatgccgct cagatcgtga tcggagccgg aatcaacatg
     2161 gccatgagaa gagtggagga gagcgtggtg aaccagggct ggatcaccct gcaggaggct
     2221 ggcatcaacc tggaccggaa caccctggcc gccatgctga tcagagagct gagagccgct
     2281 ctggagctgt tcgagcagga gggactggct ccttacctga gcagatggga gaagctggac
     2341 aacttcatca acagacctgt gaagctgatc atcggcgaca aggaaatctt cggcatctcc
     2401 agaggaatcg acaagcaggg agctctgctg ctggagcagg acggaatcat caagccctgg
     2461 atgggcggag aaatctccct gagaagcgca gagaagggat ccgtcgacgg agccggtgct
     2521 ggcgcatacc cctacgacgt gcccgactac gcaggctccg cagttccagc ggtagaaatc
     2581 acccacacct tctgggaaag gaacctgccc tctgtttctg gcctgctgaa gatcattgga
     2641 ttttccacat cagtaactgc cctggggttt gtgctgtaca aatacaagct cctgccacgg
     2701 tcttgacccg gggatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa
     2761 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2821 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2881 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2941 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3001 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3061 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3121 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3181 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3241 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3301 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3361 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3421 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3481 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3541 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3601 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3661 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3721 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3781 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3841 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3901 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3961 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4021 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4081 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4141 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4201 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4261 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4321 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4381 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4441 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4501 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4561 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4621 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4681 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4741 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4801 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4861 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4921 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4981 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5041 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5101 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5161 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5221 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5281 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5341 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5821 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6061 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6121 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6181 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6241 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6301 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6361 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6421 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6481 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6541 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6601 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6661 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6721 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6781 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6841 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6901 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6961 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7021 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7081 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7141 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
