LOCUS Exported 5216 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of your protein of interest in fusion with HA at the N-terminus. The tag is cleavable by TEV protease.. ACCESSION . VERSION . KEYWORDS pKK-HA-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5216) 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 5216) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5216 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 13..392 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 393..596 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 547..567 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 598..616 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 619..637 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 647..671 /label=LNCX /note="Human CMV promoter, forward primer" CDS 771..797 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 804..824 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 865..885 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(946..963) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 952..1176 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1309..1328) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 1460..1507 /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 1515..2537 /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 2667..2788 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2704..2723) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2758..2777 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(2837..2853) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2837..2853) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2850..2872) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2861..2877 /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(2885..2915) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2930..2951 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3068..3085) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3239..3827) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3319..3338) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3998..4858) /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 4621..4640 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4859..4963) /gene="bla" /label=AmpR promoter primer_bind complement(5038..5057) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 61 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 121 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 181 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 241 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 301 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 361 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 421 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 481 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 541 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc 601 ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga 661 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg 721 accgatccag cctccggact ctggcgttta aacttaagct tggtaccatg tacccctacg 781 acgtgcccga ctacgctgga tccgaaaacc tgtacttcca aggaaccggt ggtcacctgt 841 acaatcgatc ctgcaggagc tagcgagaat ttgtattttc agggtgatat ccagcacagt 901 ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 961 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 1021 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 1081 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 1141 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 1201 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 1261 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 1321 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 1381 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 1441 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag 1501 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg 1561 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt 1621 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt 1681 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag 1741 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg 1801 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg 1861 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg 1921 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc 1981 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg 2041 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg 2101 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact 2161 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc 2221 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg 2281 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga 2341 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg 2401 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct 2461 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc 2521 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa 2581 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 2641 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 2701 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 2761 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 2821 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 2881 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 2941 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 3001 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 3061 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 3121 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 3181 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 3241 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 3301 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 3361 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 3421 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 3481 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 3541 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 3601 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 3661 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 3721 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 3781 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 3841 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 3901 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 3961 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 4021 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 4081 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 4141 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 4201 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 4261 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 4321 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 4381 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 4441 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 4501 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 4561 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 4621 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 4681 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 4741 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 4801 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 4861 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 4921 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 4981 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac 5041 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt 5101 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg 5161 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggcca //