LOCUS Exported 6576 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Protein expression/GST pull-down. ACCESSION . VERSION . KEYWORDS TBXTA-c024 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6576) TITLE Opher Gileadi SGC expression vectors REFERENCE 2 (bases 1 to 6576) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6576 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 92..109 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 116..769 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK" primer_bind 727..749 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 797..817 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1441..1458 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1511..1529) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1525..1572 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1609..2064 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1696..1715) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1906..1927 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2156..2971) /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 2879..2898 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2963..2982 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3093..3681 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3582..3601 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3835..3852 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3867..4006 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3932..3951) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4092..4114 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4108..4299) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5108..6190) /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 6152..6171 /label=LacI-R /note="LacI, reverse primer" promoter complement(6191..6268) /gene="lacI" /label=lacI promoter primer_bind 6474..6493 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctatgtc 121 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga 181 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg 241 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga 301 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt 361 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat 421 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt 481 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata 541 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt 601 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg 661 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc 721 tttgcagggc tggcaagcca cgtttggtgg tgccgaccat cctccaaaat cgagctcagg 781 tgtagatctg ggtaccgaga acctgtactt ccaatccatg gaactgcgtg tgggtctgga 841 agaaagcgaa ctgtggctgc gctttaaaga gctgaccaac gaaatgatcg tgaccaaaaa 901 cggccgccgc atgtttcctg tgctgaaagt taacgtgagc ggcctggacc cgaacgccat 961 gtacagcttc ctgctggatt tcgttgccgc agataaccac cgctggaagt atgtgaacgg 1021 cgaatgggtg ccgggtggca aaccggaacc gcaggcacct agctgcgtgt atatccaccc 1081 ggatagccct aacttcggtg cccattggat gaaagccccg gtgagcttca gcaaggtgaa 1141 gctgaccaac aaactgaacg gcggcggcca aatcatgctg aacagcttac ataaatatga 1201 acctcgcatc cacattgtgc gtgtgggcga cccgcaacgt atgatcacca gccattgctt 1261 cccggagaca cagttcattg ccgttaccgc ctaccagaac gaggaaatta ccgcactgaa 1321 aattaaatac aacccttttg caaaagcctt cttagatgcc aaagagcgca gcgactgaca 1381 gtaaaggtgg atacggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag 1441 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg 1501 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg 1561 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta 1621 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 1681 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 1741 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 1801 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 1861 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 1921 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 1981 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 2041 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2101 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga 2161 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 2221 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 2281 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 2341 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 2401 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat 2461 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 2521 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 2581 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 2641 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 2701 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 2761 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 2821 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 2881 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag 2941 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 3001 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 3241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 3361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 3661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 3721 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 3781 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 3841 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc 3901 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 3961 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc 4021 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 4081 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 4141 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg 4201 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa 4261 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg 4321 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt 4381 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg 4441 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga 4501 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt 4561 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca 4621 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat 4681 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga 4741 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc 4801 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 4861 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 4921 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 4981 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 5041 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc 5101 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 5161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca 5221 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca 5281 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg 5341 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa 5401 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 5461 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 5521 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 5581 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca 5641 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt 5701 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 5761 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 5821 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 5881 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 5941 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 6001 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 6061 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 6121 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 6181 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 6241 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 6301 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat 6361 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc 6421 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg 6481 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt 6541 ccggcgtaga ggatcgagat ctcgatcccg cgaaat //