LOCUS Exported 7292 bp ds-DNA circular SYN 02-OCT-2017 DEFINITION Vector for in vivo biotinylation of bacterial-expressed fusion protein. Clone GOI in-frame into Sal1 and Sac1 sites. Grow plasmid in NEB Shuffle T7 Express competent bateria (NEB #C3029). Treat growing bacteria with 0.5 mM IPTG and 50 uM biotin (2.5 mM stock in 10 mM Bicine, ph 8.3) to induce expression of BirA and promote biotinylation within AviTag region of fusion protein. Best results were obtained for induction period between 3 and 5 h. MBP fusion protein can be purified with amylose resin (NEB) and proteolytically removed with Factor Xa (NEB). Cleaved protein can be separated from MBP using Ni or Co beads. ACCESSION . VERSION . KEYWORDS pMal-T-Avi-HisBirA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7292) AUTHORS Jon Backstrom TITLE Direct Submission JOURNAL Exported Tuesday, Oct 17, 2017 from SnapGene 3.1.3 http://www.snapgene.com COMMENT pMal MBP plasmid purchased from NEB. BirA in pET28a (w400-2) was a gift from Eric Campeau (Addgene plasmid # 26624). FEATURES Location/Qualifiers source 1..7292 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 3..80 /gene="lacI (mutant)" /note="lacIq promoter" /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 81..1163 /codon_start=1 /gene="lacI" /product="lac repressor" /note="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" misc_feature 1406..1434 /note="tac promoter" /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 1442..1458 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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)." CDS 1528..2628 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /note="MBP" /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" misc_feature 2329..2331 /note="sm" /note="Silent mutation (sm) added in PCR primer (for making dSac) to decrease Tm of hairpin." misc_feature 2634..2639 /note="dSac" /note="Silent mutation to remove Sac1 site: Sac1 site moved to MCS" CDS 2677..2688 /codon_start=1 /product="Factor Xa recognition and cleavage site" /note="Factor Xa site" /translation="IEGR" misc_feature 2695..2750 /note="MCS" /note="Tested with Sal1/Sac1 insert: Sal1 - spacer for efficient cleavage with Factor Xa Sac1 - removes MBP stop codon" CDS 2751..2870 /codon_start=1 /note="TEV-AviTag-HIS" /translation="LEPTTEDLYFQSDNDGSLGLNDIFEAQKIEWHGSHHHHHH" misc_feature 2805..2846 /note="AviTag" misc_feature 2832..2834 /note="biotinylation site" CDS 2853..2870 /codon_start=1 /product="6xHis affinity tag" /note="(+)6xHis" /translation="HHHHHH" terminator 2914..3000 /gene="Escherichia coli rrnB" /note="rrnB T1 terminator" /note="transcription terminator T1 from the E. coli rrnB gene" terminator 3092..3119 /note="rrnB T2 terminator" /note="transcription terminator T2 from the E. coli rrnB gene" promoter 3147..3237 /gene="bla" /note="AmpR promoter" CDS 3238..4098 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 4186..4774 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" promoter 5043..5061 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 5062..5086 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /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 5101..5123 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 5130..6092 /codon_start=1 /gene="birA" /product="E. coli biotin ligase" /note="BirA" /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" terminator 6215..6262 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(6608..6799) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg 901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag 1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1501 gcaattgacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg 1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa tgccgccagt 2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 2461 ttggtgaaag atccgcgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg 2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagttct 2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ttcacatatg 2701 tccatgggcg gccgcgatat cgtcgacaag aggtgatatc gatcggagct ctcgagccaa 2761 ccactgagga tctgtacttt cagagcgata acgatggatc cctaggactg aacgacatct 2821 tcgaagctca gaaaatcgaa tggcacggat cccatcatca ccatcaccac tagaattcga 2881 ttacaaggat gacgacgata agtaaataag cttcaaataa aacgaaaggc tcagtcgaaa 2941 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat 3001 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc 3061 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt 3121 gcgtttctac aaactctttc ggtccgttgt ttatttttct aaatacattc aaatatgtat 3181 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 3241 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 3301 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 3361 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 3421 gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 3481 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 3541 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 3601 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 3661 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 3721 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 3781 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 3841 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 3901 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 3961 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 4021 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 4081 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgatttc 4141 cttaggactg agcgtcaacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4201 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4261 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4321 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4381 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4441 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4501 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 4561 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 4621 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 4681 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 4741 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 4801 gttcctggcc ttttgctggc cttttgctca catgttccgg taccgcgccc aacagtcccc 4861 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc 4921 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg 4981 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa 5041 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat 5101 tttgtttaac tttaagaagg agatatacca tgaaggataa caccgtgcca ctgaaattga 5161 ttgccctgtt agcgaacggt gaatttcact ctggcgagca gttgggtgaa acgctgggaa 5221 tgagccgggc ggctattaat aaacacattc agacactgcg tgactggggc gttgatgtct 5281 ttaccgttcc gggtaaagga tacagcctgc ctgagcctat ccagttactt aatgctaaac 5341 agatattggg tcagctggat ggcggtagtg tagccgtgct gccagtgatt gactccacga 5401 atcagtacct tcttgatcgt atcggagagc ttaaatcggg cgatgcttgc attgcagaat 5461 accagcaggc tggccgtggt cgccggggtc ggaaatggtt ttcgcctttt ggcgcaaact 5521 tatatttgtc gatgttctgg cgtctggaac aaggcccggc ggcggcgatt ggtttaagtc 5581 tggttatcgg tatcgtgatg gcggaagtat tacgcaagct gggtgcagat aaagttcgtg 5641 ttaaatggcc taatgacctc tatctgcagg atcgcaagct ggcaggcatt ctggtggagc 5701 tgactggcaa aactggcgat gcggcgcaaa tagtcattgg agccgggatc aacatggcaa 5761 tgcgccgtgt tgaagagagt gtcgttaatc aggggtggat cacgctgcag gaagcgggga 5821 tcaatctcga tcgtaatacg ttggcggcca tgctaatacg tgaattacgt gctgcgttgg 5881 aactcttcga acaagaagga ttggcacctt atctgtcgcg ctgggaaaag ctggataatt 5941 ttattaatcg cccagtgaaa cttatcattg gtgataaaga aatatttggc atttcacgcg 6001 gaatagacaa acagggggct ttattacttg agcaggatgg aataataaaa ccctggatgg 6061 gcggtgaaat atccctgcgt agtgcagaaa aaggtaagct ttgatgagga ctagtgcggc 6121 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 6181 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 6241 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatgct agcacatgtt 6301 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 6361 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 6421 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatataagg 6481 tgcactgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 6541 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 6601 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 6661 catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 6721 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 6781 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 6841 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 6901 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 6961 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 7021 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 7081 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 7141 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 7201 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 7261 cccgtggcca ggacccaacg ctgcccgaaa tt //