LOCUS pET28a(+)-MBP-TE 6768 bp ds-DNA circular SYN 15-APR-2021 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6768) AUTHORS Trial User TITLE Direct Submission JOURNAL Exported Thursday, Jun 24, 2021 from SnapGene Viewer 5.1.7 to Vector NTI(R) format https://www.snapgene.com COMMENT VNTDATE|946857600| COMMENT VNTDBDATE|946857600| COMMENT VNTNAME|pET28a(+)-MBP-TEV-Sebm1| COMMENT VNTAUTHORNAME|Trial User| FEATURES Location/Qualifiers primer 106..125 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" /note="sequence: GGTGATGTCGGCGATATAGG" primer 209..228 /label=T7 /note="T7 promoter, forward primer" /note="sequence: TAATACGACTCACTATAGGG" 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 /label=RBS /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 CDS 335..352 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site 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." regulatory 1322..1331 /regulatory_class="other" /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer 1439..1462 /label=MBP-F /note="Maltose binding protein, forward primer" /note="sequence: GATGAAGCCCTGAAAGACGCGCAG" CDS 1481..1499 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site CDS 1505..1834 /codon_start=1 /label=Sebm1 CDS 1841..1858 /codon_start=1 /product="6xHis affinity tag" /label=6xHis primer complement(1911..1929) /label=T7 Term /note="T7 terminator, reverse primer" /note="sequence: GCTAGTTATTGCTCAGCGG" terminator 1925..1972 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2009..2464 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer complement(2096..2115) /label=F1ori-R /note="F1 origin, reverse primer" /note="sequence: AGGGAAGAAAGCGAAAGGAG" primer 2306..2327 /label=F1ori-F /note="F1 origin, forward primer" /note="sequence: GTGGACTCTTGTTCCAAACTGG" CDS complement(2556..3371) /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" primer 3279..3298 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" /note="sequence: ATCGCGAGCCCATTTATACC" primer 3363..3382 /label=pENTR-R /note="pENTR vectors, reverse primer" /note="sequence: ATGGCTCATAACACCCCTTG" rep_origin 3493..4081 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer 3982..4001 /label=pBR322ori-F /note="pBR322 origin, forward primer" /note="sequence: GGGAAACGCCTGGTATCTTT" primer 4235..4252 /label=L4440 /note="L4440 vector, forward primer" /note="sequence: AGCGAGTCAGTGAGCGAG" misc_feature 4267..4406 /label=bom /note="basis of mobility region from pBR322" primer complement(4332..4351) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" /note="sequence: CGGCATCAGAGCAGATTGTA" primer 4492..4514 /label=pGEX 3' /note="pGEX vectors, reverse primer" /note="sequence: CCGGGAGCTGCATGTGTCAGAGG" CDS complement(4508..4699) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5508..6590) /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 6552..6571 /label=LacI-R /note="LacI, reverse primer" /note="sequence: GGCATACTCTGCGACATCGT" promoter complement(6591..6668) /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 atccatggca ctggcagcaa gcctgaatgc agcagatatt accgcagcgc tggcagcatg 1561 tagcggtgtt gataccttta aacacaaaga tttctttggc aaagttggcc tgagcgcaaa 1621 aagcgcagat gatatcaaaa atgccttcaa agtgatcgac caggataaaa gcggtttcat 1681 cgaagaagaa gaactgaaac tgttcctgca gaattttagc gcaaccgcac gtgcactgac 1741 cgaagcagaa accaccgcat ttctgaaagc cggtgattca gatggtgatg gtatgattgg 1801 tatggatgaa tttgcagcca tggtgaaagg ttaactcgag caccaccacc accaccactg 1861 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca 1921 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg 1981 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg 2041 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 2101 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 2161 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 2221 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 2281 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 2341 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 2401 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca 2461 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2521 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa 2581 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 2641 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 2701 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 2761 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt 2821 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 2881 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 2941 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc 3001 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 3061 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 3121 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 3181 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 3241 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 3301 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga 3361 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat 3421 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 3481 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 3541 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 3601 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt 3661 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 3721 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 3781 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 3841 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 3901 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 3961 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 4021 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 4081 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 4141 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 4201 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 4261 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg 4321 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 4381 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 4441 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 4501 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 4561 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 4621 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 4681 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 4741 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 4801 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 4861 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 4921 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 4981 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 5041 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 5101 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 5161 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 5221 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 5281 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 5341 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 5401 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 5461 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 5521 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 5581 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 5641 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 5701 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 5761 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 5821 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 5881 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 5941 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 6001 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 6061 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 6121 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 6181 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 6241 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 6301 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 6361 gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 6421 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 6481 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 6541 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 6601 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 6661 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 6721 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatg //