LOCUS Exported 7134 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS JB96 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7134) 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 7134) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7134 /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 1483..2448 /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 2479..2505 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2725..2742) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2731..2955 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3001..3429 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3088..3107) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3298..3319 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3438..3458) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3443..3772 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3623..3758 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3685..3704 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3839..4633 /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(3893..3912) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4503..4522 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4807..4928 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4844..4863) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4898..4917 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4977..4993) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4977..4993) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4990..5012) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5001..5017 /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(5025..5055) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5070..5091 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5208..5225) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5379..5967) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5459..5478) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6138..6998) /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 6761..6780 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6999..7103) /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 gtatgactga gtacaaactg gtggtggttg gagcaggtgg tgttgggaaa 961 aacgcactga caatccagct aatccagaac cactttgtag atgaatatga tcccaccata 1021 gaggattctt acagaaaaca agtggttata gatggtgaaa cctgtttgtt ggacatactg 1081 gatacagctg gacaagaaga gtacagtgcc atgagagacc aatacatgag gacaggcgaa 1141 ggcttcctct gtgtatttgc catcaataat agcaagtcat ttgcggatat taacctctac 1201 agggagcaga ttaagcgagt aaaagactcg gatgatgtac ctatggtgct agtgggaaac 1261 aagtgtgatt tgccaacaag gacagttgat acaaaacaag cccacgaact ggccaagagt 1321 tacgggattc cattcattga aacctcagcc aagaccagac agggtgttga agatgctttt 1381 tacacactgg taagagaaat acgccagtac cgaatgaaaa aactcaacag cagtgatgat 1441 gggactcagg gtccgcggga attcggagcc ggagctggta ccatggacaa ggacaacacc 1501 gtgcccctga agctgatcgc cctgctggcc aacggcgagt tccactctgg cgagcagctg 1561 ggagagaccc tgggaatgag cagagccgcc atcaacaagc acatccagac actgagagac 1621 tggggagtgg acgtgttcac cgtgcctggc aagggctaca gcctgcctga gcctatccag 1681 ctgctgaacg ccaagcagat cctgggacag ctggatggcg gaagcgtggc cgtgctgcct 1741 gtgatcgact ccaccaatca gtacctgctg gacagaatcg gagagctgaa gtccggcgac 1801 gcctgcatcg ccgagtacca gcaggctggc agaggaggca gaggacggaa gtggttcagc 1861 ccattcggag ccaacctgta cctgtccatg ttctggagac tggagcaggg acctgctgct 1921 gccatcggac tgagtctggt gatcggaatc gtgatggccg aggtgctgag aaagctggga 1981 gccgacaagg tgagagtgaa gtggcctaat gacctgtacc tccaggaccg caagctggct 2041 ggcatcctgg tggagctgac aggcaagaca ggcgatgccg ctcagatcgt gatcggagcc 2101 ggaatcaaca tggccatgag aagagtggag gagagcgtgg tgaaccaggg ctggatcacc 2161 ctgcaggagg ctggcatcaa cctggaccgg aacaccctgg ccgccatgct gatcagagag 2221 ctgagagccg ctctggagct gttcgagcag gagggactgg ctccttacct gagcagatgg 2281 gagaagctgg acaacttcat caacagacct gtgaagctga tcatcggcga caaggaaatc 2341 ttcggcatct ccagaggaat cgacaagcag ggagctctgc tgctggagca ggacggaatc 2401 atcaagccct ggatgggcgg agaaatctcc ctgagaagcg cagagaaggg atccgtcgac 2461 ggagccggtg ctggcgcata cccctacgac gtgcccgact acgcaggctc cgcagttcca 2521 gcggtagaaa tcacccacac cttctgggaa aggaacctgc cctctgtttc tggcctgctg 2581 aagatcattg gattttccac atcagtaact gccctggggt ttgtgctgta caaatacaag 2641 ctcctgccac ggtcttgacc cggggatatc cagcacagtg gcggccgctc gagtctagag 2701 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 2761 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 2821 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 2881 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 2941 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 3001 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3061 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 3181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 3241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 3361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 3421 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 3481 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 3541 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 3601 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 3661 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga 3721 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc 3781 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat 3841 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 3901 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 3961 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4021 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4081 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4141 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4201 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4261 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4321 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 4381 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4441 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4501 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4561 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4621 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 4681 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 4741 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 4801 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 4861 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 4921 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 4981 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 5041 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 5101 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5221 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5281 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5341 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5401 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5461 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5521 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 5581 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5641 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5701 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 5761 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 5821 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 5881 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 5941 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6001 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 6721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 6781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 6841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 6901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 6961 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7021 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7081 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc //