LOCUS Exported 7221 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION mammalian expression, proximity labelling, mitochondrial relocalisation. ACCESSION . VERSION . KEYWORDS JB19 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7221) 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 7221) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7221 /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 1570..2535 /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 2566..2592 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(2812..2829) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2818..3042 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3088..3516 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3175..3194) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3385..3406 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3525..3545) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3530..3859 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3710..3845 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3772..3791 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3926..4720 /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(3980..3999) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4590..4609 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4894..5015 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4931..4950) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4985..5004 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5064..5080) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5064..5080) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5077..5099) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5088..5104 /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(5112..5142) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5157..5178 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5295..5312) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5466..6054) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5546..5565) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6225..7085) /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 6848..6867 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7086..7190) /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 gtatgggcac ccgcgacgac gagtacgact atctcttcaa agttgtcctt 961 attggagatt ctggtgttgg aaagaataat ctcttgtctc gatttactcg aaatgagttt 1021 aatctcgaaa gtaagagcac cattggagta gagtttgcaa caagaagcat ccaggttgat 1081 gggaaaacaa taaaggcaca gatatgggac acggcagggc aggagcgata ccgagctata 1141 acatcagcat attatcgtgg agctgtaggt gccttactgg tttatgacat tgctaagcat 1201 ctcacatatg aaaatgtaga acgatggctg aaagaactga gagatcatgc tgatagtaac 1261 attgttatca tgcttgtggg caataagagt gatttgcgtc atctcagggc agttcctaca 1321 gatgaagcaa gagcttttgc agaaaagaat ggtttgtcat tcattgagac ttccgctcta 1381 gactctacaa atgtagaagc tgcttttcag acaattctga cagagatata ccgcattgtt 1441 tcccagaaac aaatgtcaga cagacgtgaa aatgacatgt ctccaagcaa caatgtggtt 1501 cctattcatg ttccaccaac cactgaaaac aagccaaagg tgcaggaatt cggagccgga 1561 gctggtacca tggacaagga caacaccgtg cccctgaagc tgatcgccct gctggccaac 1621 ggcgagttcc actctggcga gcagctggga gagaccctgg gaatgagcag agccgccatc 1681 aacaagcaca tccagacact gagagactgg ggagtggacg tgttcaccgt gcctggcaag 1741 ggctacagcc tgcctgagcc tatccagctg ctgaacgcca agcagatcct gggacagctg 1801 gatggcggaa gcgtggccgt gctgcctgtg atcgactcca ccaatcagta cctgctggac 1861 agaatcggag agctgaagtc cggcgacgcc tgcatcgccg agtaccagca ggctggcaga 1921 ggaggcagag gacggaagtg gttcagccca ttcggagcca acctgtacct gtccatgttc 1981 tggagactgg agcagggacc tgctgctgcc atcggactga gtctggtgat cggaatcgtg 2041 atggccgagg tgctgagaaa gctgggagcc gacaaggtga gagtgaagtg gcctaatgac 2101 ctgtacctcc aggaccgcaa gctggctggc atcctggtgg agctgacagg caagacaggc 2161 gatgccgctc agatcgtgat cggagccgga atcaacatgg ccatgagaag agtggaggag 2221 agcgtggtga accagggctg gatcaccctg caggaggctg gcatcaacct ggaccggaac 2281 accctggccg ccatgctgat cagagagctg agagccgctc tggagctgtt cgagcaggag 2341 ggactggctc cttacctgag cagatgggag aagctggaca acttcatcaa cagacctgtg 2401 aagctgatca tcggcgacaa ggaaatcttc ggcatctcca gaggaatcga caagcaggga 2461 gctctgctgc tggagcagga cggaatcatc aagccctgga tgggcggaga aatctccctg 2521 agaagcgcag agaagggatc cgtcgacgga gccggtgctg gcgcataccc ctacgacgtg 2581 cccgactacg caggctccgc agttccagcg gtagaaatca cccacacctt ctgggaaagg 2641 aacctgccct ctgtttctgg cctgctgaag atcattggat tttccacatc agtaactgcc 2701 ctggggtttg tgctgtacaa atacaagctc ctgccacggt cttgacccgg ggatatccag 2761 cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 2821 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 2881 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 2941 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 3001 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 3061 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 3121 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 3181 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3241 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3301 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 3361 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 3421 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 3481 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 3541 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3601 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3661 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 3721 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 3781 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 3841 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 3901 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 3961 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 4021 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 4081 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 4141 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 4201 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 4261 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 4321 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 4381 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 4441 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 4501 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 4561 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 4621 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 4681 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 4741 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 4801 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 4861 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 4921 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 4981 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 5041 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 5101 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 5161 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 5221 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 5281 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 5341 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5401 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5461 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5521 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 5581 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 5641 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 5701 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 5761 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5821 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5881 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 5941 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 6001 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 6061 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6121 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6181 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6241 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6301 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6361 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6421 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6481 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6541 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6601 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6661 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6721 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6781 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6841 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6901 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 6961 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7021 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7081 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 7141 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7201 cgaaaagtgc cacctgacgt c //