LOCUS Exported 8232 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pET vector for IPTG-inducible, T7-based expression of RTX bearing a C-terminal 6xHis tag. ACCESSION . VERSION . KEYWORDS pET_RTX-6xHis SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8232) AUTHORS Bhadra S, Maranhao AC, Ellington AD TITLE A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production JOURNAL bioRxiv REFERENCE 2 (bases 1 to 8232) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8232 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 86..172 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 269..355 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 452..538 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 635..721 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 818..904 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 1039..1058 /label=T7 /note="T7 promoter, forward primer" promoter 1039..1057 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1058..1082 /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 1097..1119 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" RBS 3055..3063 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 3464..3481 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3548..3566) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3562..3609 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter 3669..3773 /gene="bla" /label=AmpR promoter CDS 3774..4634 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3992..4011) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin complement(4676..5189) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4871..4892) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5083..5102 /label=F1ori-R /note="F1 origin, reverse primer" rep_origin 5300..5888 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5789..5808 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6042..6059 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(6081..6100) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6241..6263 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(6257..6448) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(7005..8087) /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 EDGTRLGVEHLVALGHQQIALLAGPLSSVSARPRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 8049..8068 /label=LacI-R /note="LacI, reverse primer" promoter complement(8088..8165) /gene="lacI" /label=lacI promoter ORIGIN 1 gttgagcacc gccgccgcaa ggaatggtgc atgccggcat gccgcccttt cgtcttcaag 61 aattaattcc caattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 121 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg 181 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata 241 aactgccagg aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg 301 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 361 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 421 ataaactgcc aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga 481 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 541 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 601 gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa 661 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa 721 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg 781 cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa aggctcagtc 841 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 901 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg 961 acgcccgcca taaactgcca ggaattgggg atcggaatta attcccggtt taaaccgggg 1021 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt 1081 cccctctaga aataattttg tttaacttta agaaggagat atacatatga ttcttgacac 1141 ggattacatc acggaagacg gcaagccggt tatccgtatt ttcaagaaag aaaacggcga 1201 attcaagatt gaatacgatc ggacatttga accgtacctg tacgctctcc tcaaggatga 1261 tagcgcaatc gaagaagtga aaaaaatcac cgcagagcgg catggcacag tggtaacagt 1321 taagcgggtc gagaaagtgc agaagaagtt cttaggccgg ccagtcgaag tatggaaatt 1381 atacttcaca catccacagg acgttccggc gatcatggat aagattcggg agcatccggc 1441 ggtaatcgat atctatgaat acgatattcc gttcgctatt cgctacctta ttgacaaagg 1501 tttagttcca atggagggtg atgaggaact taaactgtta gcattcgata tcgaaacact 1561 ttatcacgaa ggtgaagagt ttgccgaagg tccgatttta atgatctcgt acgccgatga 1621 agaaggcgca cgcgtaatta cgtggaaaaa tgtggacctc ccgtacgtag acgtagtgag 1681 cactgagcgc gagatgatta aacgtttcct tcgggtagta aaagaaaaag acccagacgt 1741 gctgattacg tataacggcg acaactttga ttttgcctat ctcaagaagc gttgcgaaaa 1801 gttaggcatt aatttcgccc tgggtcggga cggttcagag ccgaaaattc agcggatggg 1861 cgaccgcttt gctgtggagg taaaaggtcg catccatttc gatttatatc cggttatccg 1921 gcgcaccatc aacttgccga cttacacact tgaagcagtt tacgaagcgg tgttcggcca 1981 accaaaagaa aaggtttatg ccgaggagat taccaccgca tgggaaactg gcgaaaactt 2041 ggagcgggtg gctcggtatt ccatggaaga tgccaaggtg acctacgaac tgggcaaaga 2101 gtttttaccg atggaagcac aattaagccg ccttattggt cagtccctct gggatgtgtc 2161 gcgttcttca acgggcaatt tagtcgaatg gtttcttctt cggaaagcgt acgagcgtaa 2221 cgagcttgct ccaaataagc cagacgaaaa agaattggct cggcgccatc agtcacatga 2281 gggcggctac attaaggagc cagaacgggg cttgtgggag aacatcgtct accttgattt 2341 tcggtctctt tatccgtcta ttatcatcac acataacgtc tcgccagata ccctgaaccg 2401 tgaaggctgt aaagaatatg atgtggcacc acaggtcggc catcgttttt gtaaagactt 2461 cccgggcttc attccatctc ttctgggtga tttgttagaa gagcgtcaaa agatcaagaa 2521 acgtatgaaa gcgacaattg acccaattga acgcaaatta cttgattacc gtcagcgtgc 2581 aatcaagatc ctcgcgaact ctctgtacgg ttattacggc tacgcacgcg cccggtggta 2641 ttgcaaagaa tgtgcagaat cagtcattgc ttggggtcgg gagtacctga ccatgacgat 2701 taaggaaatt gaggagaaat acggtttcaa ggtcatctat agtgacacgg atggtttctt 2761 tgcaacgatt ccaggtgcgg acgcagaaac tgtaaagaaa aaggcaatgg agttcttgaa 2821 gtatattaat gcgaagttgc caggcgccct ggaattagag tacgaaggtt tttataagcg 2881 tggcctgttc gtgacaaaga agaaatacgc ggtaattgac gaggaaggca agatcacaac 2941 tcgtggcttg gaaattgttc gtcgcgattg gagcgagatc gcaaaggaga cccaagctcg 3001 tgtgttggag gccctcctga aggatggtga cgtcgaaaaa gccgtacgca tcgttaagga 3061 ggttacagag aagcttagca agtatgaggt cccaccagag aaacttgtta ttcataaaca 3121 aatcactcgc gaccttaaag actataaggc cactggtcca cacgtcgccg tagcaaagcg 3181 gcttgcggct cggggcgtca agattcggcc aggcacggtt attagttaca tcgtcctcaa 3241 aggctcaggc cggattgttg atcgcgcgat tccatttgat gaatttgatc cgacgaagca 3301 taaatatgat gcggaatatt acattgaaaa acaggttctg ccggcggtgg agcgcatctt 3361 acgtgcgttc ggctatcgca aggaggattt gcggtaccag aaaactcgtc aagtcggttt 3421 gagtgcccgt ctgaagccga aaggtaccct cgagggatcc agccatcacc atcaccatca 3481 ctgataagcg gccgcattga tccggctgct aacaaagccc gaaaggaagc tgagttggct 3541 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg 3601 ggttttttgc tgaaaggagg aactatatcc ggataactac gtcaggtggc acttttcggg 3661 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3721 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 3781 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3841 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 3901 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3961 gtttcccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 4021 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 4081 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 4141 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 4201 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4261 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 4321 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4381 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4441 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4501 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4561 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4621 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttacccc 4681 ggttgataat cagaaaagcc ccaaaaacag gaagattgta taagcaaata tttaaattgt 4741 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 4801 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt 4861 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 4921 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag 4981 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 5041 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 5101 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 5161 cgcgcttaat gcgccgctac agggcgcgta aaaggatcta ggtgaagatc ctttttgata 5221 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5281 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5341 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5401 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 5461 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5521 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5581 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5641 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5701 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5761 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5821 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 5881 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 5941 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 6001 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 6061 aagctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 6121 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 6181 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 6241 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc 6301 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt 6361 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt 6421 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca 6481 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 6541 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 6601 accagagaaa aatcactcag ggtcaatgcc agccgaacgc cagcaagacg tagcccagcg 6661 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac 6721 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga 6781 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca 6841 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc 6901 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc 6961 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc 7021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 7081 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 7141 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 7201 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 7261 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg 7321 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 7381 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 7441 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 7501 ggacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 7561 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 7621 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 7681 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 7741 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 7801 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 7861 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 7921 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 7981 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 8041 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 8101 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg 8161 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 8221 taggttgagg cc //