LOCUS Exported 5192 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Control vector for pKK series (encodes only SLIC arms (TEV-L and TEV-R)); expression without tag. Useful to design other vectors; any tag can be added.. ACCESSION . VERSION . KEYWORDS pKK-NoTag SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5192) 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 5192) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5192 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 820..838 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 869..893 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1002..1022 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1063..1083 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(1144..1161) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1150..1374 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(1507..1526) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 1658..1705 /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 1713..2735 /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 2865..2986 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2902..2921) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2956..2975 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3035..3051) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3035..3051) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3048..3070) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3059..3075 /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(3083..3113) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3128..3149 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3266..3283) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3437..4025) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3517..3536) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4196..5056) /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 4819..4838 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5057..5161) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cgaaaacctg tacttccaag 1021 gaaccggtgg tcacctgtac aatcgatcct gcaggagcta gcgagaattt gtattttcag 1081 ggtgatatcc agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat 1141 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 1201 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 1261 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 1321 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 1381 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 1441 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1501 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1561 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1621 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct 1681 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg 1741 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg 1801 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa 1861 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg 1921 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca 1981 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg 2041 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga 2101 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca 2161 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca 2221 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag 2281 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca 2341 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca 2401 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc 2461 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc 2521 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg 2581 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc 2641 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc 2701 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca 2761 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 2821 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag 2881 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 2941 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac 3001 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt 3061 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg 3121 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt 3181 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3241 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3301 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3361 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3421 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3481 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3541 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3601 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 3661 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3721 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3781 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3841 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 3901 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3961 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4021 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4081 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4141 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4201 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4261 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4321 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4381 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4441 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4501 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4561 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4621 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4681 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 4741 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4801 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4861 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4921 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4981 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5041 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5101 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5161 gcacatttcc ccgaaaagtg ccacctgacg tc //