LOCUS Exported 6258 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION APEX targeted to the mitochondrial matrix of mammalian cells. Used for proteomic mapping of mitochondria. This construct has been replaced by APEX2 (construct 49386). . ACCESSION . VERSION . KEYWORDS pcDNA3-mito-APEX SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6258) AUTHORS Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, Ting AY TITLE Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging. JOURNAL Science. 2013 Jan 31. PUBMED 23371551 REFERENCE 2 (bases 1 to 6258) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6258 /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 988..1029 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 1030..1776 /codon_start=1 /product="soybean ascorbate peroxidase, engineered to serve as a monomeric tag for electron microscopy and live-cell proteomics (Lam et al., 2015)" /label=APEX2 /note="more active than the original APEX due to the A134P mutation" /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV EVTGGPKVPFHPGREDKPEPPPEGRLPDATKGSDHLRDVFGKAMGLTDQDIVALSGGHT IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK YAADEDAFFADYAEAHQKLSELGFADA" promoter complement(1810..1828) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1811..1828) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1848..1865) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1854..2078 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2124..2552 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2211..2230) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2421..2442 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2561..2581) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2566..2896 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2747..2882 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2809..2828 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2963..3757 /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(3017..3036) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3627..3646 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3931..4052 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3968..3987) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4022..4041 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4101..4117) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4101..4117) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4114..4136) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4125..4141 /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(4149..4179) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4194..4215 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4332..4349) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4503..5091) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4583..4602) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5262..6122) /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 5885..5904 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6123..6227) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgcggcc 901 gccaccatgc tggccacccg cgtgttcagc ctggtgggca agcgcgccat cagcaccagc 961 gtgtgcgtgc gcgcccacaa ggatccaggc aagcccatcc ccaaccccct gctgggcctg 1021 gacagcaccg gaaagtctta cccaactgtg agtgctgatt accaggacgc cgttgagaag 1081 gcgaagaaga agctcagagg cttcatcgct gagaagagat gcgctcctct aatgctccgt 1141 ttggcattcc actctgctgg aacctttgac aagggcacga agaccggtgg acccttcgga 1201 accatcaagc accctgccga actggctcac agcgctaaca acggtcttga catcgctgtt 1261 aggcttttgg agccactcaa ggcggagttc cctattttga gctacgccga tttctaccag 1321 ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta aggttccatt ccaccctgga 1381 agagaggaca agcctgagcc accaccagag ggtcgcttgc ccgatgccac taagggttct 1441 gaccatttga gagatgtgtt tggcaaagct atggggctta ctgaccaaga tatcgttgct 1501 ctatctgggg gtcacactat tggagctgca cacaaggagc gttctggatt tgagggtccc 1561 tggacctcta atcctcttat tttcgacaac tcatacttca cggagttgtt gagtggtgag 1621 aaggaaggtc tccttcagct accttctgac aaggctcttt tgtctgaccc tgtattccgc 1681 cctctcgttg ataaatatgc agcggacgaa gatgccttct ttgctgatta cgctgaggct 1741 caccaaaagc tttccgagct tgggtttgct gatgcctaat gactcgagca tgcatctaga 1801 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc 1861 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 1921 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 1981 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 2041 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 2101 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2161 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 2221 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 2281 catcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 2341 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 2401 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 2461 ctcggtctat tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa 2521 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 2581 tgtggaaagt ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta 2641 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 2701 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 2761 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 2821 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 2881 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga 2941 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 3001 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 3061 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 3121 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac 3181 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 3241 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 3301 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 3361 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 3421 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 3481 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 3541 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 3601 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 3661 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 3721 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg 3781 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat 3841 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 3901 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 3961 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4021 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4081 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4141 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 4201 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 4261 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 4321 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 4381 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 4441 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4681 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4921 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5161 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5221 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5281 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5341 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5401 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5461 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5521 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5581 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5641 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5701 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5761 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5821 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5881 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5941 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6001 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6061 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6121 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6181 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6241 aaagtgccac ctgacgtc //