LOCUS Exported 6459 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Plasmid for recombinant expression in bacteria of MBP fused to SnoopTagJr, permitting covalent linkage to DogTag by SnoopLigase. ACCESSION . VERSION . KEYWORDS pET28a-SnoopTagJr-MBP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6459) AUTHORS Buldun CM, Jean JX, Bedford MR, Howarth M TITLE SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation. JOURNAL J Am Chem Soc. 2018 Feb 28;140(8):3008-3018. doi: 10.1021/jacs.7b13237. Epub 2018 Feb 14. PUBMED 29402082 REFERENCE 2 (bases 1 to 6459) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6459 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 958..977 /label=LacI-R /note="LacI, reverse primer" promoter complement(997..1074) /gene="lacI" /label=lacI promoter primer_bind 1280..1299 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1383..1402 /label=T7 /note="T7 promoter, forward primer" promoter 1383..1401 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1402..1426 /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 1441..1463 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1482..1499 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1572..2669 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" primer_bind 2643..2666 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2706..2723 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2776..2794) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2790..2837 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2874..3329 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2961..2980) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3171..3192 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3421..4236) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 4144..4163 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4228..4247 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4358..4946 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4847..4866 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5100..5117 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5132..5271 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5197..5216) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5357..5379 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5373..5564) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6373..996) /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 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac 61 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa 121 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta 181 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc 241 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag 301 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat 361 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga 421 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg 481 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg 541 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc 601 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt 661 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct 721 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag 781 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc 841 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt 901 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc 961 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc 1021 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat 1081 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc 1141 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc 1201 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc 1261 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg 1321 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa 1381 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat 1441 tttgtttaac tttaagaagg agatatacca tgggcagcag ccatcaccac catcatcaca 1501 gcagcggcaa actgggcagc atcgaattca tcaaagtgaa caaaggtagt ggtgaaagtg 1561 gtgggtcagg gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct 1621 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg 1681 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc 1741 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg 1801 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg 1861 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt 1921 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata 1981 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca 2041 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg 2101 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg 2161 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct 2221 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca 2281 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac 2341 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa 2401 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca 2461 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta 2521 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt 2581 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg 2641 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gtaaaagctt gcggccgcac 2701 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg 2761 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg 2821 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc 2881 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2941 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 3001 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 3061 atggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 3121 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 3181 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 3241 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 3301 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga 3361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc 3421 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 3481 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 3541 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 3601 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 3661 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 3721 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 3781 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga 3841 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 3901 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 3961 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 4021 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 4081 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 4141 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 4201 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat 4261 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact 4321 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 4381 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 4441 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 4501 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 4561 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 4621 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 4681 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 4741 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 4801 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 4861 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 4921 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 4981 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 5041 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 5101 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 5161 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata 5221 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac 5281 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 5341 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 5401 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct 5461 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg 5521 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa 5581 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata 5641 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg 5701 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat 5761 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata 5821 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc 5881 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg 5941 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc 6001 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc 6061 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag 6121 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg 6181 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca 6241 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg 6301 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta 6361 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 6421 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtatt //