LOCUS Exported 6431 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses V5-tagged TurboID in the mammalian cytosol. ACCESSION . VERSION . KEYWORDS V5-TurboID-NES_pCDNA3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6431) AUTHORS Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, Feldman JL, Perrimon N, Ting AY TITLE Efficient proximity labeling in living cells and organisms with TurboID. JOURNAL Nat Biotechnol. 2018 Aug 20. pii: nbt.4201. doi: 10.1038/nbt.4201. PUBMED 30125270 REFERENCE 2 (bases 1 to 6431) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6431 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 381..584 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 629..648 /label=T7 /note="T7 promoter, forward primer" promoter 629..647 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 667..676 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 676..717 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 724..1680 /codon_start=1 /product="mutant of the E. coli biotin ligase BioID engineered for improved catalytic efficiency (Branon et al., 2018)" /label=TurboID /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK" CDS 1687..1713 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" promoter complement(1750..1768) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(1751..1768) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1788..1805) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1794..2018 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2064..2492 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2151..2170) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2361..2382 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2501..2521) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2506..2835 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2686..2821 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2748..2767 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2902..3696 /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(2956..2975) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3566..3585 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3870..3991 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3907..3926) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3961..3980 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4040..4056) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4040..4056) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4053..4075) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4064..4080 /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(4088..4118) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4133..4154 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4271..4288) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4442..5030) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4522..4541) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5201..6061) /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 5824..5843 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6062..6166) /gene="bla" /label=AmpR promoter primer_bind complement(6241..6260) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt 661 gcggccgcca ccatgggcaa gcccatcccc aaccccctgc tgggcctgga cagcaccgct 721 agcaaagaca atactgtgcc tctgaagctg atcgctctcc tggctaatgg cgagttccat 781 agtggcgaac agctgggaga aaccctgggc atgtccaggg ccgctatcaa caagcacatt 841 cagactctgc gcgactgggg cgtggacgtg ttcaccgtgc ccggaaaggg ctactctctg 901 cccgagccta tcccgctgct gaacgctaaa cagattctgg gacagctgga cggcgggagc 961 gtggcagtcc tgcctgtggt cgactccacc aatcagtacc tgctggatcg aatcggcgag 1021 ctgaagagtg gggatgcttg cattgcagaa tatcagcagg cagggagagg aagcagaggg 1081 aggaaatggt tctctccttt tggagctaac ctgtacctga gtatgttttg gcgcctgaag 1141 cggggaccag cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg 1201 cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca atgacctgta tctgcaggat 1261 agaaagctgg caggcatcct ggtggagctg gccggaataa caggcgatgc tgcacagatc 1321 gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag 1381 ggctggatca cactgcagga agcagggatt aacctggaca ggaatactct ggccgctacg 1441 ctgatccgag agctgcgggc agccctggaa ctgttcgagc aggaaggcct ggctccatat 1501 ctgccacggt gggagaagct ggataacttc atcaatagac ccgtgaagct gatcattggg 1561 gacaaagaga ttttcgggat tagccggggg attgataaac agggagccct gctgctggaa 1621 caggacggag ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag 1681 ctgcagctgc ctcccctgga gcgcctgacc ctggactaat agctcgagca tgcatctaga 1741 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc 1801 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 1861 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 1921 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 1981 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 2041 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 2461 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 2521 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 2581 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 2641 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 2701 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 2761 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 2821 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag 2881 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 2941 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 3001 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 3061 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 3121 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 3181 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 3241 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 3301 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 3361 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 3421 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 3481 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 3541 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 3601 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 3661 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga 3721 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg 3781 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg 3841 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca 3901 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3961 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct 4021 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 4081 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 4141 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 4201 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 4261 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 4321 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4381 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4441 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4561 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4621 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4681 ccaagctggg ctgtgtgcac aaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4741 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4801 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4861 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 4921 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4981 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 5041 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 5101 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 5161 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 5221 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 5281 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 5341 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 5401 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 5461 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 5521 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 5581 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5641 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5701 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5761 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 5821 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5881 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5941 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 6001 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 6061 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 6121 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 6181 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag 6241 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga 6301 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa 6361 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag 6421 atatacgcgt t //