LOCUS       Exported                7206 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  mammalian expression, proximity labelling, mitochondrial 
            relocalisation.
ACCESSION   .
VERSION     .
KEYWORDS    JB93
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7206)
  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 7206)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7206
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             1555..2520
                     /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             2551..2577
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2797..2814)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2803..3027
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3073..3501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3160..3179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3370..3391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3510..3530)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3515..3844
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3695..3830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3757..3776
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3911..4705
                     /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(3965..3984)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4575..4594
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4879..5000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4916..4935)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4970..4989
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5049..5065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5049..5065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5062..5084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5073..5089
                     /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(5097..5127)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5142..5163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5280..5297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5451..6039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5531..5550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6210..7070)
                     /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     6833..6852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7071..7175)
                     /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 gtatgacctc taggaagaaa gtgttgctga aggttatcat cctgggagat
      961 tctggagtcg ggaagacatc actcatgaac cagtatgtga ataagaaatt cagcaatcag
     1021 tacaaagcca caataggagc tgactttctg accaaggagg tgatggtgga tgacaggcta
     1081 gtcacaatgc agatatggga cacagcagga ctggaacggt tccagtctct cggtgtggcc
     1141 ttctacagag gtgcagactg ctgcgttctg gtatttgatg tgactgcccc caacacattc
     1201 aaaaccctag atagctggag agatgagttt ctcatccagg ccagtccccg agatcctgaa
     1261 aacttcccat ttgttgtgtt gggaaacaag attgacctcg aaaacagaca agtggccaca
     1321 aagcgggcac aggcctggtg ctacagcaaa aacaacattc cctactttga gaccagtgcc
     1381 aaggaggcca tcaacgtgga gcaggcgttc cagacgattg cacggaatgc acttaagcag
     1441 gaaacggagg tggagctgta caacgaattt cctgaaccta tcaaactgga caagaatgac
     1501 cgggccaagg cctcggcaga aagcccgcgg gaattcggag ccggagctgg taccatggac
     1561 aaggacaaca ccgtgcccct gaagctgatc gccctgctgg ccaacggcga gttccactct
     1621 ggcgagcagc tgggagagac cctgggaatg agcagagccg ccatcaacaa gcacatccag
     1681 acactgagag actggggagt ggacgtgttc accgtgcctg gcaagggcta cagcctgcct
     1741 gagcctatcc agctgctgaa cgccaagcag atcctgggac agctggatgg cggaagcgtg
     1801 gccgtgctgc ctgtgatcga ctccaccaat cagtacctgc tggacagaat cggagagctg
     1861 aagtccggcg acgcctgcat cgccgagtac cagcaggctg gcagaggagg cagaggacgg
     1921 aagtggttca gcccattcgg agccaacctg tacctgtcca tgttctggag actggagcag
     1981 ggacctgctg ctgccatcgg actgagtctg gtgatcggaa tcgtgatggc cgaggtgctg
     2041 agaaagctgg gagccgacaa ggtgagagtg aagtggccta atgacctgta cctccaggac
     2101 cgcaagctgg ctggcatcct ggtggagctg acaggcaaga caggcgatgc cgctcagatc
     2161 gtgatcggag ccggaatcaa catggccatg agaagagtgg aggagagcgt ggtgaaccag
     2221 ggctggatca ccctgcagga ggctggcatc aacctggacc ggaacaccct ggccgccatg
     2281 ctgatcagag agctgagagc cgctctggag ctgttcgagc aggagggact ggctccttac
     2341 ctgagcagat gggagaagct ggacaacttc atcaacagac ctgtgaagct gatcatcggc
     2401 gacaaggaaa tcttcggcat ctccagagga atcgacaagc agggagctct gctgctggag
     2461 caggacggaa tcatcaagcc ctggatgggc ggagaaatct ccctgagaag cgcagagaag
     2521 ggatccgtcg acggagccgg tgctggcgca tacccctacg acgtgcccga ctacgcaggc
     2581 tccgcagttc cagcggtaga aatcacccac accttctggg aaaggaacct gccctctgtt
     2641 tctggcctgc tgaagatcat tggattttcc acatcagtaa ctgccctggg gtttgtgctg
     2701 tacaaataca agctcctgcc acggtcttga cccggggata tccagcacag tggcggccgc
     2761 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2821 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2881 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2941 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3001 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3061 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3121 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3181 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3241 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3301 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3361 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3421 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3481 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3601 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3661 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3781 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3841 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     3901 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3961 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4021 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4081 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4141 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4201 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4261 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4321 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4381 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4441 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4501 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4561 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4621 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4681 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4741 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4801 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4861 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4921 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4981 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5041 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5101 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5161 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5221 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5281 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5341 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5401 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5461 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5521 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5581 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5641 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5701 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5761 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5821 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5881 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5941 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6001 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6061 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6121 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6181 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6241 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6301 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6361 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6421 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6481 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6541 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6601 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6661 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6721 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6781 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6841 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6901 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6961 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7021 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7081 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7141 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7201 gacgtc
//
