LOCUS Exported 8384 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression plasmid for SARS-CoV-2 surface glycoprotein (Spike protein). ACCESSION . VERSION . KEYWORDS pGBW-m4136461 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8384) TITLE SARS-CoV-2 protein E. coli expression constructs, tagged, for protein expression and/or purification REFERENCE 2 (bases 1 to 8384) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8384 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 20..39 /label=T7 /note="T7 promoter, forward primer" promoter 20..38 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 39..63 /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 78..100 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 111..128 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 141..1238 /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 1095..1104 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1212..1235 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1251..1271 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" terminator 5238..5269 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" promoter 5270..5372 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 5373..6233 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5591..5610) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" CDS 6234..6425 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" terminator 6449..6476 /label=T7Te terminator /note="phage T7 early transcription terminator" rep_origin complement(6488..7075) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6568..6587) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 7097..7126 /label=T3Te terminator /note="phage T3 early transcription terminator" CDS complement(7212..8294) /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 8256..8275 /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 61 tcccctctag aaataatttt gtttaacttt aagaaggaga ctcttctatg caccatcatc 121 accatcatgg tggttctggt aaaatcgaag aaggtaaact ggtaatctgg attaacggcg 181 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta 241 aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg 301 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg 361 gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct 421 gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat 481 cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg 541 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac 601 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg 661 gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct 721 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag 781 aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca 841 acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac 901 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag 961 agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta 1021 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag 1081 atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc 1141 cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc 1201 gtcagactgt cgatgaagcc ctgaaagacg cgcagactgg tggttctggt gagaacctgt 1261 actttcaaag ctttgtcttc ctggtactcc ttccgttggt gtccagtcaa tgcgttaacc 1321 tgaccacgag aactcagctg ccacccgcgt ataccaattc tttcactagg ggtgtttact 1381 acccggataa agtatttcgg tcgagcgttt tacattctac acaggacctg ttcctgcctt 1441 tcttctcaaa cgtgacttgg tttcacgcca ttcacgttag cggcaccaac gggaccaagc 1501 gcttcgacaa cccggtcctg ccgttcaatg atggtgtgta ctttgcatct actgagaaaa 1561 gcaacatcat acgcggctgg atcttcggaa cgactctgga ctccaaaacc cagtctctcc 1621 tgatcgtgaa caatgctact aacgtagtta ttaaggtatg tgaatttcaa ttctgcaacg 1681 atccgttctt gggcgtatat taccataaaa acaacaaatc ttggatggaa tccgaatttc 1741 gtgtttattc cagcgctaat aattgcactt tcgagtacgt tagccagcct ttcctgatgg 1801 acctggaagg caaacagggt aacttcaaaa acctgcgtga atttgttttc aagaacatcg 1861 atggttactt taaaatctat tctaagcaca ccccaattaa cctggttcgt gacctgccgc 1921 agggtttctc ggctttggag ccgctggtag atcttccgat cgggattaac atcacccgtt 1981 ttcaaaccct gctggcgctg caccgttcat acctgacacc gggtgacagt tctagcggct 2041 ggacagcagg cgctgcggcc tactatgtgg gttacctgca accgcgcacc ttcctactca 2101 aatacaacga aaatggcacg atcaccgacg ccgtcgattg tgcacttgac cctctgagcg 2161 aaactaaatg cactctgaaa tcctttacgg tcgaaaaagg tatctatcag acctccaact 2221 tccgcgttca gcctactgaa agtatcgtac gtttcccgaa cattactaac ctgtgtccct 2281 ttggagaggt ttttaacgct accaggttcg cgtctgtgta tgcttggaac cgtaagcgaa 2341 tctcaaactg cgtagcggat tacagcgtcc tgtataactc tgcaagcttt tcgactttca 2401 aatgctacgg cgtgtctcca accaagttga acgatctatg tttcaccaac gtctacgctg 2461 attctttcgt tatccgtggt gacgaggttc gtcagattgc accgggccag acaggtaaaa 2521 tcgcggatta caattataaa ttaccggacg acttcacggg ctgcgtaata gcttggaact 2581 ctaacaatct ggatagcaaa gttggcggta actacaacta cttatatcga ctgttccgca 2641 aaagcaacct gaagcccttc gaacgcgaca tctccactga aatttaccag gccggttcga 2701 ccccgtgcaa cggtgtagaa ggttttaatt gctacttccc gttgcagtcc tatggctttc 2761 agccgaccaa cggcgttggc tatcagcctt accgtgttgt ggtgctctcc tttgaactgc 2821 tgcatgcccc tgcaaccgta tgtggcccga aaaaatccac taacttagtg aaaaacaaat 2881 gcgtaaactt taatttcaat ggtctgacag ggacgggcgt cctgactgaa tctaacaaaa 2941 agttcctgcc attccaacag tttggtcgcg atattgctga cactaccgat gcagttcgtg 3001 acccgcagac gctggaaatc ctggatatca ccccgtgttc tttcggtggc gttagcgtga 3061 tcactccggg tacaaacacc tccaaccagg tggctgtcct gtaccaggac gttaactgca 3121 cggaagtacc ggtggcaatt cacgctgatc agctgactcc gacgtggcgt gtctactcta 3181 ccggttcgaa tgttttccaa acgcgcgctg gctgcctgat tggtgctgaa catgtaaaca 3241 attcttacga gtgcgacatc cccataggag cgggtatctg tgcgagctac cagactcaga 3301 ccaacagccc tcgtcgcgcc cgttccgtgg caagccagtc aatcatcgcg tatacgatga 3361 gcctgggtgc cgagaactcg gttgcataca gtaacaatag cattgctatc ccaactaact 3421 tcacaatttc agttaccaca gaaatccttc cggtatctat gactaaaact tccgtggatt 3481 gcaccatgta catttgcggc gacagcactg agtgttccaa tcttttactg caatatggta 3541 gtttttgcac gcagctgaac cgtgccctga ccggaatcgc ggttgaacag gataaaaaca 3601 cccaggaagt tttcgctcaa gtaaaacaga tttataagac cccgcccatc aaagacttcg 3661 gcggttttaa tttcagccaa atcctccccg acccgtctaa gccaagcaaa cgctcattca 3721 tcgaagacct gctgtttaac aaagtgactc tggcggacgc tggcttcatc aagcaatacg 3781 gagactgtct aggggatatt gcagctcgtg acctcatttg cgcgcagaag ttcaacggct 3841 taactgttct gccgccgctg ctgaccgatg agatgatcgc tcagtacaca tccgctctgc 3901 tggcaggtac catcacctct gggtggacgt ttggcgcggg tgccgctctc cagattccat 3961 tcgccatgca aatggcttat cgctttaatg gaatcggcgt aactcagaac gttttgtacg 4021 aaaaccagaa actgatagct aaccaattca attctgcgat tggcaaaatc caggactctc 4081 tgagctccac tgcaagcgct ctcggcaaac tgcaagacgt tgtcaaccag aacgcgcagg 4141 cactgaacac cctggtaaaa cagctgtcaa gcaacttcgg tgctatcagt tcagttttaa 4201 atgatatcct gtcgcggctg gataaagttg aagccgaagt tcaaatcgac cgtctgatta 4261 ccggtcgtct gcaatctctg caaacttatg taacccagca gctgatcaga gctgccgaga 4321 ttcgcgctag cgcaaacttg gcggcgacaa aaatgtcaga atgcgtactt ggccagtcta 4381 aacgtgtgga cttctgcggt aaaggttacc acctgatgtc gtttccgcag tcggcccctc 4441 atggtgtagt tttcctacac gtgacttacg tcccggccca ggaaaagaac tttaccactg 4501 ctccagccat ctgccacgat ggtaaagctc atttcccgcg tgaaggcgtc ttcgtttcca 4561 acggcactca ctggttcgtg acgcagcgta acttctacga gccgcagatc attactactg 4621 ataacacctt tgtttcgggt aactgcgatg tcgtgatcgg catcgttaat aacactgttt 4681 acgacccgct gcaaccggaa ctggattcct ttaaagaaga actggataaa tattttaaaa 4741 accacacctc tccagatgta gacctgggag acattagtgg cataaatgcg tccgttgtta 4801 acattcagaa agaaatcgac cgcctgaacg aggtcgcaaa gaatctgaac gagtctctga 4861 ttgacctgca agagctgggt aagtatgagc agtacattaa atggccttgg tacatctggc 4921 tgggcttcat cgcaggtttg atcgccatcg tcatggtgac tattatgctc tgctgtatga 4981 cgtcttgctg ctcctgcctg aaaggttgtt gctcttgcgg gtcgtgttgc aagtttgacg 5041 aagatgacag tgaaccggtt ctgaaaggcg ttaaacttca ttacacctaa agagaatata 5101 aaaagccaga ttattaatcc ggctttttta ttatttaggt tctgttaagt aactgaaccc 5161 aatgtcgtta gtgacgctta cctcttaaga ggtcactgac ctaacagctt gactccagcg 5221 taactggact gcaatcaact caaaagcctc cgaccggagg cttttgactt gatcggcacg 5281 taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt 5341 tatcgagatt ttcaggagct aaggaagcta aaatgagtat tcaacatttc cgtgtcgccc 5401 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 5461 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 5521 acagcggtaa gatccttgag agtttacgcc ccgaagaacg ttttccaatg atgagcactt 5581 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 5641 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 5701 atctcacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 5761 acactgcggc caacttactt ctggcaacga tcggaggacc gaaggagcta accgcttttt 5821 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 5881 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 5941 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 6001 aggcggataa agttgcagga tcacttctgc gctcggccct cccggctggc tggtttattg 6061 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6121 atggtaagcc ctcccgcatc gtagttatct acacgacggg gagtcaggca actatggatg 6181 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taagtgacca 6241 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc 6301 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg 6361 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc 6421 tctgatgagg gcccaaatgt aatcacctgg ctcaccttcg ggtgggcctt tctgcgttgc 6481 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga tgctcaagtc 6541 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 6601 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 6661 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 6721 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6781 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 6841 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6901 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 6961 cagttacctc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7021 cggtggtctt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 7081 tcctttgatt ttctaccgaa gaaaggccca cccgtgaagg tgagccagtg actttctgct 7141 atggaggtca ggtatgattt aaatggtcag tgcggcgcgc caacaaaaaa cccctagccc 7201 cccgttttat atcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 7261 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt 7321 ttcaccagtg agactggcaa cagctgattg cccttcaccg cctggccctg agagagttgc 7381 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac 7441 ggcgggatat aacatgagct atcttcggta tcgtcgtatc ccactaccga gatatccgca 7501 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg 7561 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa 7621 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg 7681 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct 7741 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg 7801 tcctcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac 7861 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag 7921 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct 7981 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga 8041 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg 8101 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc 8161 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg 8221 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac 8281 gttactggtt tcatattcac caccctgaat tgactgtctt ccgggcgcta tcatgccata 8341 ccgcgaaagg ttttgcgcca ttcgatacta gtagcggccg gatg //