LOCUS Exported 7152 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS JB54 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7152) 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 7152) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7152 /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 1501..2466 /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 2497..2523 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2743..2760) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2749..2973 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3019..3447 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3106..3125) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3316..3337 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3456..3476) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3461..3790 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3641..3776 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3703..3722 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3857..4651 /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(3911..3930) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4521..4540 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4825..4946 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4862..4881) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4916..4935 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4995..5011) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4995..5011) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5008..5030) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5019..5035 /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(5043..5073) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5088..5109 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5226..5243) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5397..5985) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5477..5496) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6156..7016) /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 6779..6798 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7017..7121) /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 gtatgcaggc catcaagtgt gtggtggtgg gagacggagc tgtaggtaaa 961 aattgcctac tgatcagtta cacaaccaat gcatttcctg gagaatatat ccctactgtc 1021 tttgacaatt attctgccaa tgttatggta gatggaaaac cggtgaatct gggcttatgg 1081 gatacagctg gacaagaaga ttatgacaga ttacgccccc tatcctatcc gcaaacagat 1141 gtgttcttaa tttgcttttc ccttgtgagt cctgcatcat ttgaaaatgt ccgtgcaaag 1201 tggtatcctg aggtgcggca ccactgtccc aacactccca tcatcctagt gggaactaaa 1261 cttgatctta gggatgataa agacacgatc gagaaactga aggagaagaa gctgactccc 1321 atcacctatc cgcagggtct agccatggct aaggagattg gtgctgtaaa atacctggag 1381 tgctcggcgc tcacacagcg aggcctcaag acagtgtttg acgaagcgat ccgagcagtc 1441 ctctgcccgc ctcccgtgaa gaagaggaag agaaaagaat tcggagccgg agctggtacc 1501 atggacaagg acaacaccgt gcccctgaag ctgatcgccc tgctggccaa cggcgagttc 1561 cactctggcg agcagctggg agagaccctg ggaatgagca gagccgccat caacaagcac 1621 atccagacac tgagagactg gggagtggac gtgttcaccg tgcctggcaa gggctacagc 1681 ctgcctgagc ctatccagct gctgaacgcc aagcagatcc tgggacagct ggatggcgga 1741 agcgtggccg tgctgcctgt gatcgactcc accaatcagt acctgctgga cagaatcgga 1801 gagctgaagt ccggcgacgc ctgcatcgcc gagtaccagc aggctggcag aggaggcaga 1861 ggacggaagt ggttcagccc attcggagcc aacctgtacc tgtccatgtt ctggagactg 1921 gagcagggac ctgctgctgc catcggactg agtctggtga tcggaatcgt gatggccgag 1981 gtgctgagaa agctgggagc cgacaaggtg agagtgaagt ggcctaatga cctgtacctc 2041 caggaccgca agctggctgg catcctggtg gagctgacag gcaagacagg cgatgccgct 2101 cagatcgtga tcggagccgg aatcaacatg gccatgagaa gagtggagga gagcgtggtg 2161 aaccagggct ggatcaccct gcaggaggct ggcatcaacc tggaccggaa caccctggcc 2221 gccatgctga tcagagagct gagagccgct ctggagctgt tcgagcagga gggactggct 2281 ccttacctga gcagatggga gaagctggac aacttcatca acagacctgt gaagctgatc 2341 atcggcgaca aggaaatctt cggcatctcc agaggaatcg acaagcaggg agctctgctg 2401 ctggagcagg acggaatcat caagccctgg atgggcggag aaatctccct gagaagcgca 2461 gagaagggat ccgtcgacgg agccggtgct ggcgcatacc cctacgacgt gcccgactac 2521 gcaggctccg cagttccagc ggtagaaatc acccacacct tctgggaaag gaacctgccc 2581 tctgtttctg gcctgctgaa gatcattgga ttttccacat cagtaactgc cctggggttt 2641 gtgctgtaca aatacaagct cctgccacgg tcttgacccg gggatatcca gcacagtggc 2701 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 2761 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2821 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2881 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 2941 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg 3001 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 3061 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 3121 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 3181 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 3241 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 3301 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 3361 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 3421 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg 3481 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 3541 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3601 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3661 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 3721 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 3781 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg 3841 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg 3901 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc 3961 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg 4021 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt 4081 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg 4141 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat 4201 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca 4261 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca 4321 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa 4381 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa 4441 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc 4501 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga 4561 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc 4621 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac 4681 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg 4741 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 4801 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 4861 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 4921 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 4981 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 5041 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 5101 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 5161 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 5221 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5281 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 5341 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 5401 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 5461 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 5521 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5581 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 5641 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 5701 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 5761 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 5821 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 5881 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 5941 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 6001 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 6061 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 6121 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 6181 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 6241 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 6301 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 6361 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 6421 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 6481 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 6541 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 6601 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 6661 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 6721 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 6781 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 6841 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 6901 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 6961 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 7021 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 7081 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7141 ccacctgacg tc //