LOCUS Exported 6386 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses His6-tagged TurboID in bacteria. ACCESSION . VERSION . KEYWORDS TurboID-His6_pET21a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6386) 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 6386) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6386 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 940..959 /label=LacI-R /note="LacI, reverse primer" promoter complement(979..1056) /gene="lacI" /label=lacI promoter primer_bind 1262..1281 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1365..1384 /label=T7 /note="T7 promoter, forward primer" promoter 1365..1383 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1384..1408 /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)." RBS 1423..1445 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1453..1485 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 1501..2460 /codon_start=1 /product="mutant of the E. coli biotin ligase BioID engineered for improved catalytic efficiency (Branon et al., 2018)" /label=TurboID /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD WGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSG DACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKL GADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGW ITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGD KEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK" CDS 2482..2499 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2552..2570) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2566..2613 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2650..3105 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2737..2756) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2947..2968 /label=F1ori-F /note="F1 origin, forward primer" promoter 3131..3235 /gene="bla" /label=AmpR promoter CDS 3236..4096 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3454..3473) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4267..4855 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4756..4775 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5009..5026 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5041..5180 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5106..5125) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5266..5288 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5282..5473) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6282..978) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" ORIGIN 1 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag 61 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt 121 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc 181 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc 241 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg 301 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg 361 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc 421 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt 481 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa 541 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg 601 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca 661 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc 721 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc 781 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta 841 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc 901 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta 961 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc 1021 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat 1081 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg 1141 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca 1201 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat 1261 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca 1321 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat 1381 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa 1441 ggagatatac atatggctag catgactggt ggacagcaaa tgggtcgcgg atccgaattc 1501 atgaaagaca atactgtgcc tctgaagctg atcgctctcc tggctaatgg cgagttccat 1561 agtggcgaac agctgggaga aaccctgggc atgtccaggg ccgctatcaa caagcacatt 1621 cagactctgc gcgactgggg cgtggacgtg ttcaccgtgc ccggaaaggg ctactctctg 1681 cccgagccta tcccgctgct gaacgctaaa cagattctgg gacagctgga cggcgggagc 1741 gtggcagtcc tgcctgtggt cgactccacc aatcagtacc tgctggatcg aatcggcgag 1801 ctgaagagtg gggatgcttg cattgcagaa tatcagcagg cagggagagg aagcagaggg 1861 aggaaatggt tctctccttt tggagctaac ctgtacctga gtatgttttg gcgcctgaag 1921 cggggaccag cagcaatcgg cctgggcccg gtcatcggaa ttgtcatggc agaagcgctg 1981 cgaaagctgg gagcagacaa ggtgcgagtc aaatggccca atgacctgta tctgcaggat 2041 agaaagctgg caggcatcct ggtggagctg gccggaataa caggcgatgc tgcacagatc 2101 gtcattggcg ccgggattaa cgtggctatg aggcgcgtgg aggaaagcgt ggtcaatcag 2161 ggctggatca cactgcagga agcagggatt aacctggaca ggaatactct ggccgctacg 2221 ctgatccgag agctgcgggc agccctggaa ctgttcgagc aggaaggcct ggctccatat 2281 ctgccacggt gggagaagct ggataacttc atcaatagac ccgtgaagct gatcattggg 2341 gacaaagaga ttttcgggat tagccggggg attgataaac agggagccct gctgctggaa 2401 caggacggag ttatcaaacc ctggatgggc ggagaaatca gtctgcggtc tgccgaaaag 2461 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa 2521 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 2581 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg 2641 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2701 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2761 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2821 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 2881 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2941 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 3001 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3061 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg 3121 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 3181 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 3241 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3301 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 3361 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 3421 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 3481 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 3541 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 3601 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 3661 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 3721 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc 3781 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 3841 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 3901 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 3961 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 4021 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 4081 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 4141 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4201 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4261 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4321 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4381 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 4441 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 4501 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 4561 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 4621 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 4681 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 4741 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 4801 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 4861 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 4921 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 4981 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 5041 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact 5101 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 5161 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 5221 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 5281 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 5341 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 5401 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 5461 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac 5521 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 5581 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 5641 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 5701 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 5761 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 5821 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 5881 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg 5941 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag 6001 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca 6061 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct 6121 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc 6181 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg 6241 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt 6301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 6361 tgcgtattgg gcgccagggt ggtttt //