LOCUS Exported 6565 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses V5-tagged TurboID on the mammalian ER membrane. ACCESSION . VERSION . KEYWORDS C1(1-29)-TurboID-V5_pCDNA3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6565) 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 6565) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6565 /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" regulatory 900..909 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1065..2021 /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 2028..2069 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" promoter complement(2117..2135) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2118..2135) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2155..2172) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2161..2385 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2431..2859 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2518..2537) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2728..2749 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2868..2888) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2873..3203 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3054..3189 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3116..3135 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3270..4064 /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(3324..3343) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3934..3953 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4238..4359 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4275..4294) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4329..4348 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4408..4424) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4408..4424) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4421..4443) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4432..4448 /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(4456..4486) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4501..4522 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4639..4656) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4810..5398) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4890..4909) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5569..6429) /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 6192..6211 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6430..6534) /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 gcttgggccg 901 ccaccatgga ccctgtggtc gtgctggggc tgtgcctgag ctgtctgctg ctgctgagcc 961 tgtggaagca gtcctacgga ggcgggggat caggaagcgg cggccgcgga tcaggaagcg 1021 gatcaggaag cggatcagga agcggatcag gaagcggatc aggaaaagac aatactgtgc 1081 ctctgaagct gatcgctctc ctggctaatg gcgagttcca tagtggcgaa cagctgggag 1141 aaaccctggg catgtccagg gccgctatca acaagcacat tcagactctg cgcgactggg 1201 gcgtggacgt gttcaccgtg cccggaaagg gctactctct gcccgagcct atcccgctgc 1261 tgaacgctaa acagattctg ggacagctgg acggcgggag cgtggcagtc ctgcctgtgg 1321 tcgactccac caatcagtac ctgctggatc gaatcggcga gctgaagagt ggggatgctt 1381 gcattgcaga atatcagcag gcagggagag gaagcagagg gaggaaatgg ttctctcctt 1441 ttggagctaa cctgtacctg agtatgtttt ggcgcctgaa gcggggacca gcagcaatcg 1501 gcctgggccc ggtcatcgga attgtcatgg cagaagcgct gcgaaagctg ggagcagaca 1561 aggtgcgagt caaatggccc aatgacctgt atctgcagga tagaaagctg gcaggcatcc 1621 tggtggagct ggccggaata acaggcgatg ctgcacagat cgtcattggc gccgggatta 1681 acgtggctat gaggcgcgtg gaggaaagcg tggtcaatca gggctggatc acactgcagg 1741 aagcagggat taacctggac aggaatactc tggccgctac gctgatccga gagctgcggg 1801 cagccctgga actgttcgag caggaaggcc tggctccata tctgccacgg tgggagaagc 1861 tggataactt catcaataga cccgtgaagc tgatcattgg ggacaaagag attttcggga 1921 ttagccgggg gattgataaa cagggagccc tgctgctgga acaggacgga gttatcaaac 1981 cctggatggg cggagaaatc agtctgcggt ctgccgaaaa ggctagcggc aagcccatcc 2041 ccaaccccct gctgggcctg gacagcacct aatgaggatc cggcaagccc atccccaacc 2101 ccctgctggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga 2161 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 2221 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 2281 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 2341 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 2401 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 2461 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 2521 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 2581 atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 2641 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 2701 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 2761 accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt 2821 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 2881 gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc 2941 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3001 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 3061 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 3121 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 3181 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 3241 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 3301 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 3361 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 3421 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg 3481 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 3541 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 3601 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 3661 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 3721 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 3781 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 3841 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 3901 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 3961 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 4021 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 4081 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 4141 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 4201 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 4261 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 4321 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 4381 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4441 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4501 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4561 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4621 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4681 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4741 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4801 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4861 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4921 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4981 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 5041 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5101 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5161 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5221 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 5281 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5341 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5401 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5461 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5521 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 5581 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5641 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5701 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 5761 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 5821 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5881 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5941 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6001 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6061 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6121 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6181 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6241 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 6301 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 6361 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6421 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6481 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6541 tccccgaaaa gtgccacctg acgtc //