LOCUS Exported 7233 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS pY49 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7233) 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 7233) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7233 /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 1582..2547 /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 2578..2604 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2824..2841) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2830..3054 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3100..3528 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3187..3206) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3397..3418 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3537..3557) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3542..3871 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3722..3857 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3784..3803 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3938..4732 /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(3992..4011) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4602..4621 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4906..5027 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4943..4962) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4997..5016 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5076..5092) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5076..5092) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5089..5111) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5100..5116 /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(5124..5154) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5169..5190 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5307..5324) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5478..6066) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5558..5577) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6237..7097) /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 6860..6879 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7098..7202) /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 gtaggacaat ggggacccgg gacgacgagt acgactacct attcaaagtg 961 gtgctcatcg gggactcagg cgtgggcaag aacaacctgc tgtcgcgctt cacccgcaac 1021 gagttcaacc tggagagcaa gagcaccatc ggcgtggagt tcgccacccg cagcatccag 1081 gtggacggca agaccatcaa ggcgcagatc tgggacaccg ctggccagga gcgctaccgc 1141 gccatcacct ccgcgtacta ccgtggtgca gtgggcgccc tgctggtgta cgacatcgcc 1201 aagcacctga cctatgagaa cgtggagcgc tggctgaagg agctgcggga ccacgcagac 1261 agcaacatcg tcatcatgct ggtgggcaac aagagtgacc tgcgccacct gcgggctgtg 1321 cccactgacg aggcccgcgc cttcgcagaa aagaacaact tgtccttcat cgagaccgca 1381 gccttggatt ccactaacgt agaggaagca ttcaagaaca tcctcacaga gatctaccgc 1441 atcgtgtcac agaaacagat cgcagaccgt gctgcccacg acgagtcccc ggggaacaac 1501 gtggtggaca tcagcgtgcc gcccaccacg gacggacaga agcccaacaa gctgcaggaa 1561 ttcggagccg gagctggtac catggacaag gacaacaccg tgcccctgaa gctgatcgcc 1621 ctgctggcca acggcgagtt ccactctggc gagcagctgg gagagaccct gggaatgagc 1681 agagccgcca tcaacaagca catccagaca ctgagagact ggggagtgga cgtgttcacc 1741 gtgcctggca agggctacag cctgcctgag cctatccagc tgctgaacgc caagcagatc 1801 ctgggacagc tggatggcgg aagcgtggcc gtgctgcctg tgatcgactc caccaatcag 1861 tacctgctgg acagaatcgg agagctgaag tccggcgacg cctgcatcgc cgagtaccag 1921 caggctggca gaggaggcag aggacggaag tggttcagcc cattcggagc caacctgtac 1981 ctgtccatgt tctggagact ggagcaggga cctgctgctg ccatcggact gagtctggtg 2041 atcggaatcg tgatggccga ggtgctgaga aagctgggag ccgacaaggt gagagtgaag 2101 tggcctaatg acctgtacct ccaggaccgc aagctggctg gcatcctggt ggagctgaca 2161 ggcaagacag gcgatgccgc tcagatcgtg atcggagccg gaatcaacat ggccatgaga 2221 agagtggagg agagcgtggt gaaccagggc tggatcaccc tgcaggaggc tggcatcaac 2281 ctggaccgga acaccctggc cgccatgctg atcagagagc tgagagccgc tctggagctg 2341 ttcgagcagg agggactggc tccttacctg agcagatggg agaagctgga caacttcatc 2401 aacagacctg tgaagctgat catcggcgac aaggaaatct tcggcatctc cagaggaatc 2461 gacaagcagg gagctctgct gctggagcag gacggaatca tcaagccctg gatgggcgga 2521 gaaatctccc tgagaagcgc agagaaggga tccgtcgacg gagccggtgc tggcgcatac 2581 ccctacgacg tgcccgacta cgcaggctcc gcagttccag cggtagaaat cacccacacc 2641 ttctgggaaa ggaacctgcc ctctgtttct ggcctgctga agatcattgg attttccaca 2701 tcagtaactg ccctggggtt tgtgctgtac aaatacaagc tcctgccacg gtcttgaccc 2761 ggggatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat 2821 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2881 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2941 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 3001 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 3061 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 3121 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 3181 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3241 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3301 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3361 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3421 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 3481 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 3541 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3601 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 3661 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 3721 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 3781 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 3841 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 3901 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 3961 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 4021 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 4081 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 4141 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 4201 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 4261 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 4321 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 4381 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 4441 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 4501 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 4561 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 4621 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 4681 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 4741 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 4801 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 4861 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 4921 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 4981 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 5041 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 5101 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 5161 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 5221 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 5281 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 5341 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 5401 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5461 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5521 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5581 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5641 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 5701 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5761 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5821 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5881 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 5941 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 6001 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 6061 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 6121 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 6181 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 6241 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 6301 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 6361 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 6421 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 6481 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 6541 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 6601 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 6661 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 6721 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 6781 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 6841 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 6901 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 6961 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 7021 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 7081 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 7141 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 7201 cgcacatttc cccgaaaagt gccacctgac gtc //