LOCUS Exported 6484 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET28a-SpyTagMBP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6484) AUTHORS Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M TITLE Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. JOURNAL Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7. Epub 2012 Feb 24. PUBMED 22366317 REFERENCE 2 (bases 1 to 6484) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6484 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 65..1162 /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 1136..1159 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1199..1216 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1269..1287) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1283..1330 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1367..1822 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1454..1473) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1664..1685 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1915..2730) /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 2638..2657 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2722..2741 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2852..3440 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3341..3360 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3594..3611 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3626..3768 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3694..3713) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3854..3876 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3870..4061) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4870..5952) /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 5914..5933 /label=LacI-R /note="LacI, reverse primer" promoter complement(5953..6030) /gene="lacI" /label=lacI promoter primer_bind 6236..6255 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6339..6358 /label=T7 /note="T7 promoter, forward primer" promoter 6339..6357 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6358..6382 /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 6397..6419 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6438..6455 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 6465..6482 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" ORIGIN 1 tatgggagcc cacatcgtga tggtggacgc ctacaagccg acgaagggta gtggtgaaag 61 tggtaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg 121 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca 181 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat 241 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat 301 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta 361 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa 421 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact 481 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc 541 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa 601 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat 661 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa 721 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa 781 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt 841 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt 901 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct 961 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc 1021 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt 1081 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga 1141 agccctgaaa gacgcgcaga ctaattcgag ctcgtaaaag cttgcggccg cactcgagca 1201 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 1261 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 1321 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc 1381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 1441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 1501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 1561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 1621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 1681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 1741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 1801 aaaatattaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct 1861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa 1921 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata 1981 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat 2041 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa 2101 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc 2161 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt 2221 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg 2281 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa 2341 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc 2401 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg 2461 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct 2521 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc 2581 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc 2641 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga 2701 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc 2761 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 2821 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 2881 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 2941 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 3001 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 3061 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 3121 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 3181 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 3241 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 3301 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 3361 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 3421 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 3481 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 3541 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 3601 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg 3661 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 3721 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg 3781 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 3841 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 3901 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc 3961 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata 4021 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg 4081 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg 4141 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta 4201 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca 4261 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg 4321 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt 4381 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt 4441 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac 4501 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc 4561 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt 4621 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg 4681 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc 4741 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag 4801 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt 4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt 4981 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag 5041 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg 5101 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc 5161 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc 5221 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc 5281 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag 5341 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa 5401 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc 5461 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc 5521 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt 5581 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc 5641 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga 5701 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc 5761 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 5821 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt 5881 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt 5941 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc 6001 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact 6061 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga 6121 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac 6181 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga 6241 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc 6301 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga 6361 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat 6421 ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca 6481 gcca //