LOCUS Exported 6287 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of your protein of interest in fusion with MBP at the N-terminus (cleavable by TEV). The maltose binding protein tag allows convenient purification of proteins (PMID: 22442034, 19935684).. ACCESSION . VERSION . KEYWORDS pKK-MBP-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6287) 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 6287) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6287 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(32..51) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 223..602 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 603..806 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 757..777 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 808..826 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 829..847 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 857..881 /label=LNCX /note="Human CMV promoter, forward primer" CDS 978..2078 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" regulatory 1935..1944 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 2052..2075 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 2085..2105 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2146..2166 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(2227..2244) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2233..2457 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2590..2609) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2741..2788 /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 2796..3818 /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 3948..4069 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3985..4004) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4039..4058 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4118..4134) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4118..4134) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4131..4153) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4142..4158 /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(4166..4196) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4211..4232 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4349..4366) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4520..5108) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4600..4619) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5279..6139) /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 5902..5921 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6140..6244) /gene="bla" /label=AmpR promoter ORIGIN 1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 781 ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg 841 atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga cgccatccac 901 gctgttttga cctccataga agacaccggg accgatccag cctccggact ctggcgttta 961 aacttaagct tggtaccatg aaaactgaag aaggtaaact ggtaatctgg attaacggcg 1021 ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta 1081 aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg 1141 gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg 1201 gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct 1261 gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat 1321 cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg 1381 cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac 1441 cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg 1501 gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct 1561 tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag 1621 aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca 1681 acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac 1741 catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag 1801 agctggcgaa agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta 1861 ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag 1921 atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc 1981 cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc 2041 gtcagactgt cgatgaagcc ctgaaagacg cgcagactgg atccgaaaac ctgtacttcc 2101 aaggaaccgg tggtcacctg tacaatcgat cctgcaggag ctagcgagaa tttgtatttt 2161 cagggtgata tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct 2221 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 2281 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 2341 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 2401 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 2461 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 2521 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2581 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2641 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2701 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt 2761 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt 2821 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg 2881 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg 2941 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg 3001 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt 3061 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg 3121 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga 3181 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt 3241 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg 3301 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg 3361 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc 3421 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg 3481 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg 3541 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg 3601 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc 3661 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg 3721 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa 3781 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt 3841 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 3901 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg 3961 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 4021 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta 4081 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 4141 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 4201 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 4261 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 4321 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 4381 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 4441 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 4501 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 4561 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 4621 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 4681 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 4741 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 4801 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 4861 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 4921 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 4981 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 5041 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 5101 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 5161 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 5221 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 5281 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 5341 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 5401 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 5461 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 5521 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 5581 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 5641 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 5701 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 5761 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 5821 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 5881 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 5941 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 6001 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 6061 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 6121 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 6181 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 6241 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcggga //