LOCUS Exported 7218 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS JB2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7218) 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 7218) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7218 /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 1567..2532 /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 2563..2589 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2809..2826) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2815..3039 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3085..3513 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3172..3191) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3382..3403 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3522..3542) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3527..3856 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3707..3842 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3769..3788 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3923..4717 /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(3977..3996) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4587..4606 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4891..5012 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4928..4947) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4982..5001 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5061..5077) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5061..5077) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5074..5096) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5085..5101 /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(5109..5139) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5154..5175 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5292..5309) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5463..6051) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5543..5562) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6222..7082) /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 6845..6864 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7083..7187) /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 gtatggcgta cgcctatctc ttcaagtaca tcataatcgg cgacacaggt 961 gttggtaaaa actgcttatt gctacagttt acagacaaga ggtttcagcc agtgcatgac 1021 cttactattg gtgtagagtt cggtgctcga atgataacta ttgatgggaa acagataaaa 1081 cttcagatat gggatacggc agggcaagaa tcctttcgtt ccatcacaag gtcgtattac 1141 agaggtgcag caggagcttt actagtttac gatattacac ggagagatac attcaaccac 1201 ttgacaacct ggttagaaga tgcccgccag cattccaatt ccaacatggt cattatgctt 1261 attggaaata aaagtgattt agaatctaga agagaagtaa aaaaagaaga aggtgaagct 1321 tttgcacgag aacatggact catcttcatg gaaacgtctg ctaagactgc ttccaatgta 1381 gaagaggcat ttattaatac agcaaaagaa atttatgaaa aaattcaaga aggagtcttt 1441 gacattaata atgaggcaaa tggcattaaa attggccctc agcatgctgc taccaatgca 1501 acacatgcag gcaatcaggg aggacagcag gctgggggcg gcgaattcgg agccggagct 1561 ggtaccatgg acaaggacaa caccgtgccc ctgaagctga tcgccctgct ggccaacggc 1621 gagttccact ctggcgagca gctgggagag accctgggaa tgagcagagc cgccatcaac 1681 aagcacatcc agacactgag agactgggga gtggacgtgt tcaccgtgcc tggcaagggc 1741 tacagcctgc ctgagcctat ccagctgctg aacgccaagc agatcctggg acagctggat 1801 ggcggaagcg tggccgtgct gcctgtgatc gactccacca atcagtacct gctggacaga 1861 atcggagagc tgaagtccgg cgacgcctgc atcgccgagt accagcaggc tggcagagga 1921 ggcagaggac ggaagtggtt cagcccattc ggagccaacc tgtacctgtc catgttctgg 1981 agactggagc agggacctgc tgctgccatc ggactgagtc tggtgatcgg aatcgtgatg 2041 gccgaggtgc tgagaaagct gggagccgac aaggtgagag tgaagtggcc taatgacctg 2101 tacctccagg accgcaagct ggctggcatc ctggtggagc tgacaggcaa gacaggcgat 2161 gccgctcaga tcgtgatcgg agccggaatc aacatggcca tgagaagagt ggaggagagc 2221 gtggtgaacc agggctggat caccctgcag gaggctggca tcaacctgga ccggaacacc 2281 ctggccgcca tgctgatcag agagctgaga gccgctctgg agctgttcga gcaggaggga 2341 ctggctcctt acctgagcag atgggagaag ctggacaact tcatcaacag acctgtgaag 2401 ctgatcatcg gcgacaagga aatcttcggc atctccagag gaatcgacaa gcagggagct 2461 ctgctgctgg agcaggacgg aatcatcaag ccctggatgg gcggagaaat ctccctgaga 2521 agcgcagaga agggatccgt cgacggagcc ggtgctggcg cataccccta cgacgtgccc 2581 gactacgcag gctccgcagt tccagcggta gaaatcaccc acaccttctg ggaaaggaac 2641 ctgccctctg tttctggcct gctgaagatc attggatttt ccacatcagt aactgccctg 2701 gggtttgtgc tgtacaaata caagctcctg ccacggtctt gacccgggga tatccagcac 2761 agtggcggcc gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc 2821 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 2881 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 2941 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 3001 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 3061 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 3121 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 3181 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 3241 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 3301 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 3361 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 3421 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 3481 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 3541 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3601 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 3661 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 3721 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 3781 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 3841 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga 3901 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 3961 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 4021 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 4081 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac 4141 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 4201 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 4261 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 4321 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 4381 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 4441 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 4501 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 4561 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 4621 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 4681 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg 4741 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 4801 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 4861 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4921 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4981 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 5041 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 5101 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5161 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 5221 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 5401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 5461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 5521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5641 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5881 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 5941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 6001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 6061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 6181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 6241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 6301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 6361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 6421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 6481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 6541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 6601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 7021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 7081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 7201 aaagtgccac ctgacgtc //