LOCUS Exported 4853 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Used for expression of protein in E. coli as PreScission cleavable N-terminal Avi-His8-tag and thioredoxin fusions. ACCESSION . VERSION . KEYWORDS pOP3TP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4853) TITLE Hyvonen Lab pOP plasmids REFERENCE 2 (bases 1 to 4853) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4853 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 74..93 /label=T7 /note="T7 promoter, forward primer" promoter 74..92 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 92..116 /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 129..151 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 165..209 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 219..242 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" CDS 249..572 /codon_start=1 /gene="trxA" /product="E. coli thioredoxin" /label=TrxA /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL DANL" CDS 603..626 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" promoter complement(676..694) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(677..694) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(752..770) /label=T7 Term /note="T7 terminator, reverse primer" terminator 766..813 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(1049..1070) /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1220..2302) /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 2264..2283 /label=LacI-R /note="LacI, reverse primer" promoter complement(2303..2380) /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." primer_bind 2523..2542 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(2696..2715) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 2815..2837 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(2875..2893) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2961..3065 /gene="bla" /label=AmpR promoter CDS 3066..3926 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3284..3303) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4097..4685 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4586..4605 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4839..3 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 61 taatgcagct gattaatacg actcactata gggaattgtg agcggataac aattcctcta 121 gaaataattt tgtttaactt taagaaggag atatatccat gaatggactg aatgatatct 181 ttgaagcgca gaaaattgaa tggcatgaat ccggatctca tcaccatcac catcaccatc 241 acactagtat gagcgataaa attattcacc tgactgacga cagttttgac acggatgtac 301 tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa 361 tgatcgcccc gattctggat gaaatcgctg acgaatatca gggcaaactg accgttgcaa 421 aactgaacat cgatcaaaac cctggcactg cgccgaaata tggcatccgt ggtatcccga 481 ctctgctgct gttcaaaaac ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag 541 gtcagttgaa agagttcctc gacgctaacc tggcgaccgg tagtggcacc agtgggtcga 601 cactggaagt tctgtttcag ggcccaggat ccatggaatt cgcggccgcc ctaggctcga 661 gctaagcttg agtattctat agtgtcacct aaatcccagc ttgatccggc tgctaacaaa 721 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 781 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggataa 841 cctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 901 atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 961 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 1021 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 1081 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cacagcttgc 1141 atgcctgcag gtcggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 1201 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 1261 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg 1321 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag 1381 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg 1441 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga 1501 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct 1561 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt 1621 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat 1681 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg 1741 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc 1801 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa 1861 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca 1921 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt 1981 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat 2041 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg 2101 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa 2161 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc 2221 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat 2281 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc 2341 atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc gggtaccgag ctcgaattgt 2401 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2461 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2521 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2581 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ctgatgcggt 2641 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 2701 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 2761 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 2821 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 2881 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 2941 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 3001 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 3061 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 3121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 3181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 3241 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 3301 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 3361 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 3421 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 3481 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 3541 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 3601 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 3661 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 3721 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 3781 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 3841 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 3901 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 3961 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 4021 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 4081 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4141 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4201 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 4261 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4321 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4381 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4441 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4501 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4561 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4621 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4681 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4741 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4801 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agc //