LOCUS Exported 5678 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expressed 7+ sortase A variant in E.coli. ACCESSION . VERSION . KEYWORDS pET30b-7+-SrtA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5678) AUTHORS Jeong HJ, Abhiraman GC, Story CM, Ingram JR, Dougan SK TITLE Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling. JOURNAL PLoS One. 2017 Dec 4;12(12):e0189068. doi: 10.1371/journal.pone.0189068. eCollection 2017. PUBMED 29200433 REFERENCE 2 (bases 1 to 5678) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5678 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 539..556 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(609..627) /label=T7 Term /note="T7 terminator, reverse primer" terminator 623..670 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 707..1162 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(794..813) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1004..1025 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1254..2069) /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 1977..1996 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2061..2080 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2191..2779 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2680..2699 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2933..2950 /label=L4440 /note="L4440 vector, forward primer" misc_feature 2965..3104 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3030..3049) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3190..3212 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3206..3397) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4206..5288) /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 5250..5269 /label=LacI-R /note="LacI, reverse primer" promoter complement(5289..5366) /gene="lacI" /label=lacI promoter primer_bind 5572..5591 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaagctaaa cctcagattc ccaaggacaa 121 gtcgaaggta gctgggtata ttgaaatccc agatgcggac attaaagaac cagtttatcc 181 aggcccggca actcgtgaac agcttaaccg tggggtatct tttgccaaag agaatcagtc 241 acttgacgac cagaatatct ctatcgctgg gcacacgttc attggacgcc cgaactatca 301 attcaccaat ctgaaggcag ccaagaaggg tagtatggtg tacttcaaag ttggtaatga 361 aactcgcaaa tataagatga ccagtattcg caatgttaaa cccactgcag tggaagtcct 421 ggatgagcag aagggcaagg ataagcagtt gactcttatt acatgcgacg atttaaaccg 481 cgaaacagga gtgtgggaaa ctcgcaaaat tttcgtggct acagaagtca aactcgagca 541 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 601 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 661 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc 721 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 781 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 841 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 901 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 961 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 1021 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 1081 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 1141 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta 1201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa 1261 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat 1321 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg 1381 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat 1441 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc 1501 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta 1561 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga 1621 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac 1681 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct 1741 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga 1801 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg 1861 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct 1921 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg 1981 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag 2041 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca 2101 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2161 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2221 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2281 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 2341 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2401 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2461 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2521 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2581 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2641 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2701 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2761 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 2821 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 2881 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 2941 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg 3001 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 3061 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa 3121 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3181 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3241 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt 3301 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc 3361 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat 3421 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta 3481 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga 3541 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg 3601 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc 3661 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg 3721 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg 3781 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca 3841 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc 3901 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga 3961 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa 4021 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa 4081 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc 4141 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt 4201 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 4261 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc 4321 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag 4381 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg 4441 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg 4501 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc 4561 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac 4621 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat 4681 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc 4741 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca 4801 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga 4861 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg 4921 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg 4981 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta 5041 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc 5101 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc 5161 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc 5221 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact 5281 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga 5341 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg 5401 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg 5461 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac 5521 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc 5581 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc 5641 ggcgtagagg atcgagatcg atctcgatcc cgcgaaat //