LOCUS Exported 5793 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expression plasmid for N-terminal his tagged S.pyogenes SrtA. ACCESSION . VERSION . KEYWORDS pET28a-SpySrtA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5793) AUTHORS Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AE, Ploegh HL TITLE Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. JOURNAL Nat Protoc. 2013 Sep;8(9):1787-99. doi: 10.1038/nprot.2013.101. Epub 2013 Aug 29. PUBMED 23989673 REFERENCE 2 (bases 1 to 5793) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5793 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 5..144 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(70..89) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 230..252 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(246..437) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(1246..2328) /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" primer_bind 2290..2309 /label=LacI-R /note="LacI, reverse primer" promoter complement(2329..2406) /gene="lacI" /label=lacI promoter primer_bind 2612..2631 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 2715..2734 /label=T7 /note="T7 promoter, forward primer" promoter 2715..2733 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 2734..2758 /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 2773..2795 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2814..2831 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 2841..2858 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 3372..3389 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3442..3460) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3456..3503 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3540..3995 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3627..3646) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3837..3858 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(4087..4902) /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 4810..4829 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4894..4913 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 5024..5612 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5513..5532 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5766..5783 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg 61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg 121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga 181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca 301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg 361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt 421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg 481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg 541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa 601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc 661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt 721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac 781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca 841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc 901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga 961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg 1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc 1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg 1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat 1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc 1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg 1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc 1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc 1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat 1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat 1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc 1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg 1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa 1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt 1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc 1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg 1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga 2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga 2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc 2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt 2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata 2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct 2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat 2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc 2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga 2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa 2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct 2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc 2761 tctagaaata attttgttta actttaagaa ggagatatac catgcgcagc agccatcatc 2821 atcatcatca cagcagcggc ctggtgccgc gcggatccgt cttgcaagca caaatggcgg 2881 ctcagcaact tcctgttata gggggcattg ccataccaga gcttggcatt aatttaccaa 2941 tttttaaagg tttaggaaat actgagctta tttatggcgc aggaacgatg aaagaagaac 3001 aagttatggg aggagaaaat aattattctc ttgccagtca tcatattttt ggaattacag 3061 gttcatctca aatgctcttt tcgccgcttg aaagagcaca aaatgggatg tccatctatt 3121 taacagataa agaaaaaatt tacgaataca tcataaaaga tgttttcacg gtagctcctg 3181 aacgcgttga tgttatcgat gatacagctg gtctcaaaga agtgacttta gtgacttgta 3241 cagatatcga agcaacagaa cgtattattg tcaaaggaga actaaaaaca gaatacgact 3301 ttgataaagc gcccgccgat gtattgaaag cttttaatca ttcttataac caagtatcta 3361 cctagctcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg 3421 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta 3481 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga 3541 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 3601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 3661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 3721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 3781 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 3841 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 3901 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 3961 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg 4021 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat 4081 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt 4141 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca 4201 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat 4261 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt 4321 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac 4381 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg 4441 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg 4501 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc 4561 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca 4621 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 4681 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt 4741 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg 4801 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa 4861 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact 4921 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt 4981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 5041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 5101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 5161 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 5221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 5281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 5341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 5401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 5461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 5521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 5581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 5641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg 5701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 5761 agcgcagcga gtcagtgagc gaggaagcgg aag //