LOCUS Exported 5589 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of your protein of interest in fusion with two IgG binding domains of Staphylococcus aureus protein A at the C-terminus (cleavable by TEV) (PMID: 3507693, 10504710). Protein purification.. ACCESSION . VERSION . KEYWORDS pKK-TEV-ProteinA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5589) AUTHORS Szczesny RJ, Kowalska K, Klosowska-Kosicka K, Chlebowski A, Owczarek EP, Warkocki Z, Kulinski TM, Adamska D, Affek K, Jedroszkowiak A, Kotrys AV, Tomecki R, Krawczyk PS, Borowski LS, Dziembowski A TITLE Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system. JOURNAL PLoS One. 2018 Mar 28;13(3):e0194887. doi: 10.1371/journal.pone.0194887. eCollection 2018. PUBMED 29590189 REFERENCE 2 (bases 1 to 5589) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5589 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(39..58) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 230..609 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 610..813 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 764..784 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 815..833 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 836..854 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 864..888 /label=LNCX /note="Human CMV promoter, forward primer" CDS 997..1017 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1058..1078 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1118..1291 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL AEAKKLNDAQAPK" CDS 1295..1465 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNGAQAPK" primer_bind complement(1536..1553) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1542..1766 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1899..1918) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2050..2097 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 2105..3127 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 3257..3378 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3294..3313) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3348..3367 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3427..3443) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3427..3443) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3440..3462) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3451..3467 /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)." promoter complement(3475..3505) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3520..3541 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3658..3675) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3829..4417) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3909..3928) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4588..5448) /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 5211..5230 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5449..5553) /gene="bla" /label=AmpR promoter ORIGIN 1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca 61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc 121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg 181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 781 cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct 841 atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc 901 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccggactctg 961 gcgtttaaac ttaagcttgg taccgagctc ggatccgaaa acctgtactt ccaaggaacc 1021 ggtggtcacc tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga 1081 tatcaaaacc gcggctcttg cgcaacacga tgaagccgtg gacaacaaat tcaacaaaga 1141 acaacaaaac gcgttctatg agatcttaca tttacctaac ttaaacgaag aacaacgaaa 1201 cgccttcatc caaagtttaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc 1261 taaaaagcta aatgatgctc aggcgccgaa agtagacaac aaattcaaca aagaacaaca 1321 aaacgcgttc tatgagatct tacatttacc taacttaaac gaagaacaac gaaacgcctt 1381 catccaaagt ttaaaagatg acccaagcca aagcgctaac cttttagcag aagctaaaaa 1441 gctaaatggt gctcaggcgc cgaaagtaga cgcgaattcc gcggggaagt caacctgact 1501 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 1561 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 1621 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 1681 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 1741 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag 1801 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 1861 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 1921 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 1981 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 2041 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt 2101 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga 2161 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga 2221 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga 2281 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat 2341 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt 2401 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga 2461 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg 2521 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta 2581 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga 2641 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg 2701 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc 2761 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc 2821 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc 2881 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga 2941 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg 3001 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg 3061 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa 3121 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 3181 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga 3241 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag 3301 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 3361 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta 3421 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 3481 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 3541 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 3601 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3661 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3721 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3781 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3841 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 3901 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 3961 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4021 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4081 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4141 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4201 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 4261 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 4321 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4381 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4441 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4501 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4561 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4621 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4681 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4741 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4801 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 4861 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 4921 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 4981 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5041 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5101 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5161 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 5221 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5281 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 5341 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5401 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5461 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5521 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5581 cgtcgacgg //