LOCUS Exported 6419 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes 3xGluGlu tagged human c-Jun for purification by nickel affinity. ACCESSION . VERSION . KEYWORDS pET-29b-3xGluGlu-c-jun-His6 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6419) AUTHORS Smolko CM, Janes KA TITLE An ultrasensitive fiveplex activity assay for cellular kinases. JOURNAL Sci Rep. 2019 Dec 19;9(1):19409. doi: 10.1038/s41598-019-55998-8. PUBMED 31857650 REFERENCE 2 (bases 1 to 6419) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6419 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 921..940 /label=LacI-R /note="LacI, reverse primer" promoter complement(960..1037) /gene="lacI" /label=lacI promoter primer_bind 1243..1262 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1350..1369 /label=T7 /note="T7 promoter, forward primer" promoter 1350..1368 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1369..1393 /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 1408..1430 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1441..1485 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" CDS 1501..1518 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1522..1539 /codon_start=1 /product="epitope tag from middle T antigen of mouse polyoma virus" /label=Glu-Glu tag /translation="EYMPME" CDS 1540..1557 /codon_start=1 /product="epitope tag from middle T antigen of mouse polyoma virus" /label=Glu-Glu tag /translation="EYMPME" CDS 1558..1575 /codon_start=1 /product="epitope tag from middle T antigen of mouse polyoma virus" /label=Glu-Glu tag /translation="EYMPME" CDS complement(1606..1617) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" CDS 2629..2646 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2699..2717) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2713..2760 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2797..3252 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2884..2903) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3094..3115 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3344..4159) /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 4067..4086 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4151..4170 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4281..4869 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4770..4789 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5023..5040 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5055..5194 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5120..5139) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5280..5302 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5296..5487) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6296..959) /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" ORIGIN 1 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 61 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 121 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg 181 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 241 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 301 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 361 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 421 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 481 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 541 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 601 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 661 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 721 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 781 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 841 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 901 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 961 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 1021 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 1081 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 1141 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca 1201 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata 1261 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag 1321 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc 1381 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg 1441 aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg acagcccaga tctgggtacc 1501 ctggtgccac gcggttccat ggagtatatg ccgatggagg agtatatgcc gatggaggag 1561 tatatgccga tggagggatc cgaattcatg actgcaaaga tggaaacgac cttctatgac 1621 gatgccctca acgcctcgtt cctcccgtcc gagagcggac cttatggcta cagtaacccc 1681 aagatcctga aacagagcat gaccctgaac ctggccgacc cagtggggag cctgaagccg 1741 cacctccgcg ccaagaactc ggacctcctc acctcgcccg acgtggggct gctcaagctg 1801 gcgtcgcccg agctggagcg cctgataatc cagtccagca acgggcacat caccaccacg 1861 ccgaccccca cccagttcct gtgccccaag aacgtgacag atgagcagga gggcttcgcc 1921 gagggcttcg tgcgcgccct ggccgaactg cacagccaga acacgctgcc cagcgtcacg 1981 tcggcggcgc agccggtcaa cggggcaggc atggtggctc ccgcggtagc ctcggtggca 2041 gggggcagcg gcagcggcgg cttcagcgcc agcctgcaca gcgagccgcc ggtctacgca 2101 aacctcagca acttcaaccc aggcgcgctg agcagcggcg gcggggcgcc ctcctacggc 2161 gcggccggcc tggcctttcc cgcgcaaccc cagcagcagc agcagccgcc gcaccacctg 2221 ccccagcaga tgcccgtgca gcacccgcgg ctgcaggccc tgaaggagga gcctcagaca 2281 gtgcccgaga tgcccggcga gacaccgccc ctgtccccca tcgacatgga gtcccaggag 2341 cggatcaagg cggagaggaa gcgcatgagg aaccgcatcg ctgcctccaa gtgccgaaaa 2401 aggaagctgg agagaatcgc ccggctggag gaaaaagtga aaaccttgaa agctcagaac 2461 tcggagctgg cgtccacggc caacatgctc agggaacagg tggcacagct taaacagaaa 2521 gtcatgaacc acgttaacag tgggtgccaa ctcatgctaa cgcagcagtt gcaaacattt 2581 ggatccgaat tcgcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac 2641 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 2701 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 2761 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa 2821 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 2881 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 2941 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3001 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 3061 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 3121 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 3181 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3241 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3301 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga 3361 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa 3421 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct 3481 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt 3541 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg 3601 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat 3661 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa 3721 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga 3781 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga 3841 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa 3901 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat 3961 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg 4021 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt 4081 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt 4141 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta 4201 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4261 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4321 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4381 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 4441 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4501 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4561 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4621 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4681 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 4741 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 4801 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 4861 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 4921 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 4981 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 5041 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 5101 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 5161 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 5221 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 5281 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg 5341 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc 5401 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt 5461 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat 5521 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga 5581 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga 5641 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc 5701 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga 5761 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca 5821 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt 5881 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat 5941 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt 6001 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag 6061 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag 6121 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac 6181 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat 6241 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact 6301 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6361 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacg //