LOCUS       sequence-254210-        7221 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 7221)
  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 7221)
  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: JB20
FEATURES             Location/Qualifiers
     source          1..7221
                     /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(1570..1923,1924..1926,1927..2535)
                     /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             2566..2592
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2812..2829)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2818..3042
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3088..3516
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3175..3194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3385..3406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3525..3545)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3530..3859
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3710..3845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3772..3791
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3926..4720
                     /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(3980..3999)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4590..4609
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4894..5015
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4931..4950)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4985..5004
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5064..5080)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5064..5080)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5077..5099)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5088..5104
                     /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(5112..5118,5119..5136,5137..5142))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5157..5178
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5295..5312)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5466..6054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5546..5565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6225..7016,7017..7085))
                     /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     6848..6867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7086..7190)
                     /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 gtatgggcac ccgcgacgac gagtacgact atctcttcaa agttgtcctt
      961 attggagatt ctggtgttgg aaagagtaat ctcttgtctc gatttactcg aaatgagttt
     1021 aatctcgaaa gtaagagcac cattggagta gagtttgcaa caagaagcat ccaggttgat
     1081 gggaaaacaa taaaggcaca gatatgggac acggcagggc tggagcgata ccgagctata
     1141 acatcagcat attatcgtgg agctgtaggt gccttactgg tttatgacat tgctaagcat
     1201 ctcacatatg aaaatgtaga acgatggctg aaagaactga gagatcatgc tgatagtaac
     1261 attgttatca tgcttgtggg caataagagt gatttgcgtc atctcagggc agttcctaca
     1321 gatgaagcaa gagcttttgc agaaaagaat ggtttgtcat tcattgagac ttccgctcta
     1381 gactctacaa atgtagaagc tgcttttcag acaattctga cagagatata ccgcattgtt
     1441 tcccagaaac aaatgtcaga cagacgtgaa aatgacatgt ctccaagcaa caatgtggtt
     1501 cctattcatg ttccaccaac cactgaaaac aagccaaagg tgcaggaatt cggagccgga
     1561 gctggtacca tggacaagga caacaccgtg cccctgaagc tgatcgccct gctggccaac
     1621 ggcgagttcc actctggcga gcagctggga gagaccctgg gaatgagcag agccgccatc
     1681 aacaagcaca tccagacact gagagactgg ggagtggacg tgttcaccgt gcctggcaag
     1741 ggctacagcc tgcctgagcc tatccagctg ctgaacgcca agcagatcct gggacagctg
     1801 gatggcggaa gcgtggccgt gctgcctgtg atcgactcca ccaatcagta cctgctggac
     1861 agaatcggag agctgaagtc cggcgacgcc tgcatcgccg agtaccagca ggctggcaga
     1921 ggaggcagag gacggaagtg gttcagccca ttcggagcca acctgtacct gtccatgttc
     1981 tggagactgg agcagggacc tgctgctgcc atcggactga gtctggtgat cggaatcgtg
     2041 atggccgagg tgctgagaaa gctgggagcc gacaaggtga gagtgaagtg gcctaatgac
     2101 ctgtacctcc aggaccgcaa gctggctggc atcctggtgg agctgacagg caagacaggc
     2161 gatgccgctc agatcgtgat cggagccgga atcaacatgg ccatgagaag agtggaggag
     2221 agcgtggtga accagggctg gatcaccctg caggaggctg gcatcaacct ggaccggaac
     2281 accctggccg ccatgctgat cagagagctg agagccgctc tggagctgtt cgagcaggag
     2341 ggactggctc cttacctgag cagatgggag aagctggaca acttcatcaa cagacctgtg
     2401 aagctgatca tcggcgacaa ggaaatcttc ggcatctcca gaggaatcga caagcaggga
     2461 gctctgctgc tggagcagga cggaatcatc aagccctgga tgggcggaga aatctccctg
     2521 agaagcgcag agaagggatc cgtcgacgga gccggtgctg gcgcataccc ctacgacgtg
     2581 cccgactacg caggctccgc agttccagcg gtagaaatca cccacacctt ctgggaaagg
     2641 aacctgccct ctgtttctgg cctgctgaag atcattggat tttccacatc agtaactgcc
     2701 ctggggtttg tgctgtacaa atacaagctc ctgccacggt cttgacccgg ggatatccag
     2761 cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     2821 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2881 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2941 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3001 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3061 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3121 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3181 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3241 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3301 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3361 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3421 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3481 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3541 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3601 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3661 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3721 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3781 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3841 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3901 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3961 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4021 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4081 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4141 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4201 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4261 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4321 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4381 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4441 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4501 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4561 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4621 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4681 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4741 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4801 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4861 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4921 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4981 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5041 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5101 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5161 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5221 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5281 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5341 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5401 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5461 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5521 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5581 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5641 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5701 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5761 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5821 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5881 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5941 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6001 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6061 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6121 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6181 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6241 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6301 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6361 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6421 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6481 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6541 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6601 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6661 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6721 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6781 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6841 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6901 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6961 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7021 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7081 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7141 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7201 cgaaaagtgc cacctgacgt c
//
