LOCUS Exported 7203 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS pO40 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7203) 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 7203) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7203 /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 1552..2517 /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 2548..2574 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2794..2811) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2800..3024 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3070..3498 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3157..3176) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3367..3388 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3507..3527) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3512..3841 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3692..3827 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3754..3773 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3908..4702 /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(3962..3981) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4572..4591 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4876..4997 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4913..4932) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4967..4986 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5046..5062) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5046..5062) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5059..5081) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5070..5086 /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(5094..5124) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5139..5160 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5277..5294) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5448..6036) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5528..5547) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6207..7067) /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 6830..6849 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7068..7172) /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 acatctttga tcaccagatt catgtatgac 1021 agttttgaca acacctatca ggcaacaatt ggcattgact ttttatcaaa aactatgtac 1081 ttggaggatc gaacagtacg attgcaatta tgggacacag caggtttgga 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 aggaggcgaa ttcggagccg gagctggtac catggacaag 1561 gacaacaccg tgcccctgaa gctgatcgcc ctgctggcca acggcgagtt ccactctggc 1621 gagcagctgg gagagaccct gggaatgagc agagccgcca tcaacaagca catccagaca 1681 ctgagagact ggggagtgga cgtgttcacc gtgcctggca agggctacag cctgcctgag 1741 cctatccagc tgctgaacgc caagcagatc ctgggacagc tggatggcgg aagcgtggcc 1801 gtgctgcctg tgatcgactc caccaatcag tacctgctgg acagaatcgg agagctgaag 1861 tccggcgacg cctgcatcgc cgagtaccag caggctggca gaggaggcag aggacggaag 1921 tggttcagcc cattcggagc caacctgtac ctgtccatgt tctggagact ggagcaggga 1981 cctgctgctg ccatcggact gagtctggtg atcggaatcg tgatggccga ggtgctgaga 2041 aagctgggag ccgacaaggt gagagtgaag tggcctaatg acctgtacct ccaggaccgc 2101 aagctggctg gcatcctggt ggagctgaca ggcaagacag gcgatgccgc tcagatcgtg 2161 atcggagccg gaatcaacat ggccatgaga agagtggagg agagcgtggt gaaccagggc 2221 tggatcaccc tgcaggaggc tggcatcaac ctggaccgga acaccctggc cgccatgctg 2281 atcagagagc tgagagccgc tctggagctg ttcgagcagg agggactggc tccttacctg 2341 agcagatggg agaagctgga caacttcatc aacagacctg tgaagctgat catcggcgac 2401 aaggaaatct tcggcatctc cagaggaatc gacaagcagg gagctctgct gctggagcag 2461 gacggaatca tcaagccctg gatgggcgga gaaatctccc tgagaagcgc agagaaggga 2521 tccgtcgacg gagccggtgc tggcgcatac ccctacgacg tgcccgacta cgcaggctcc 2581 gcagttccag cggtagaaat cacccacacc ttctgggaaa ggaacctgcc ctctgtttct 2641 ggcctgctga agatcattgg attttccaca tcagtaactg ccctggggtt tgtgctgtac 2701 aaatacaagc tcctgccacg gtcttgaccc ggggatatcc agcacagtgg cggccgctcg 2761 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc 2821 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2881 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2941 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 3001 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 3061 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 3121 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 3181 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 3241 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 3301 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3361 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 3421 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3481 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 3541 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3601 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3661 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 3721 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 3781 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 3841 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 3901 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3961 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 4021 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 4081 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 4141 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 4201 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 4261 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 4321 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 4381 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 4441 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 4501 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 4561 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 4621 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 4681 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 4741 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 4801 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 4861 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 4921 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 4981 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 5041 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5101 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 5161 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5221 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5281 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5341 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5401 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5461 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 5521 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 5581 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 5641 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5701 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5761 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5821 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5881 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5941 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6001 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6061 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6121 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6181 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6241 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6301 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6361 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6421 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6481 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 6541 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 6601 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6661 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6721 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6781 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6841 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6901 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6961 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7021 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7081 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7141 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 7201 gtc //