LOCUS Exported 7034 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION bacterial expression of MBP-TEV n-terminal fusions. ACCESSION . VERSION . KEYWORDS pET28-MBP-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7034) AUTHORS Currinn H, Guscott B, Balklava Z, Rothnie A, Wassmer T TITLE APP controls the formation of PI(3,5)P vesicles through its binding of the PIKfyve complex. JOURNAL Cell Mol Life Sci. 2015 Jul 28. PUBMED 26216398 REFERENCE 2 (bases 1 to 7034) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7034 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 106..125 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 209..228 /label=T7 /note="T7 promoter, forward primer" promoter 209..227 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 228..252 /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 267..289 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 308..325 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 335..352 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 368..1465 /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" regulatory 1322..1331 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1439..1462 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1481..1501 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2107..2124 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2177..2195) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2191..2238 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2275..2730 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2362..2381) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2572..2593 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2822..3637) /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 3545..3564 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3629..3648 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3759..4347 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4248..4267 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4501..4518 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4533..4672 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4598..4617) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4758..4780 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4774..4965) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5774..6856) /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 6818..6837 /label=LacI-R /note="LacI, reverse primer" promoter complement(6857..6934) /gene="lacI" /label=lacI promoter ORIGIN 1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa 61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat 121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta 181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg 241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg 301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc 361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa 421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga 481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga 541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga 601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg 661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa 721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga 781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg 841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat 901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct 961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa 1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag 1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt 1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga 1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc 1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc 1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc 1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga 1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg 1501 atccctcagt acaggctgct actgacagat tacttaaaga accttctaga agactctgta 1561 gatcacagag acacccaaga tgccctggct gttgtcatag aggtagctaa ccatgccaat 1621 gacaccatga agcaagggga caactttcag aagctcatgc agattcagta cagcttaagt 1681 ggacaccatg aaattgtgca acctgggcgg gtttttctta aagaaggcac cctgatgaag 1741 ctgtctcgga aagtcatgca gccccggatg tttttcctgt ttaacgatgc tcttctctac 1801 accacaccca tgcagtctgg gatgtacaaa ctgaacaaca tgctctcgct ggctggaatg 1861 aaggtcagaa aacctaccca ggaggcatat cagaacgagt taaagattga aagtgtagag 1921 cgttctttca tcctctcagc tagttctgcc gcagaaagag acgactggct ggaggccata 1981 tccagctcca tagaagagta tgccaagaaa agaatcacat tctgccccag caggagtctt 2041 gatgaggact cagaaaggaa agaagaagtt agccccctcg gggcaaaggc tccctaaccg 2101 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct 2161 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 2221 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc 2281 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2341 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2401 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2461 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2521 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 2581 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2641 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2701 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt 2821 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa 2881 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc 2941 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac 3001 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga 3061 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc 3121 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt 3181 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg 3241 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat 3301 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat 3361 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt 3421 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa 3481 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga 3541 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg 3601 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta 3661 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac 3721 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 3781 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 3841 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 3901 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 3961 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 4021 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 4081 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 4141 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 4201 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 4261 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 4321 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 4381 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 4441 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 4501 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat 4561 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat 4621 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca 4681 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4741 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4801 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc 4861 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct 4921 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta 4981 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat 5041 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc 5101 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa 5161 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat 5221 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac 5281 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc 5341 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct 5401 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg 5461 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa 5521 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc 5581 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac 5641 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt 5701 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt 5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc 5881 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt 5941 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta 6001 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg 6061 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt 6121 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa 6181 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag 6241 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg 6301 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac 6361 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata 6421 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat 6481 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg 6541 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc 6601 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa 6661 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat 6721 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct 6781 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata 6841 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca 6901 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc 6961 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca 7021 aggaatggtg catg //