LOCUS Exported 6618 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Functional studies using RNAi; rescue experiments; substituting protein for its different form. miRNA cassette with nuclear EGFP expression marker; your protein of interest with N-terminal FLAG tag.. ACCESSION . VERSION . KEYWORDS pKK-RNAi-nucEGFPmiR-FLAG-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6618) 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 6618) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6618 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(23..43) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" polyA_signal 438..486 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind 511..530 /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" ncRNA complement(602..643) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(707..733) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(740..781) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(845..871) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(878..919) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(983..1009) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" CDS complement(1055..1075) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1079..1099) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1103..1123) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1136..1852) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(1136..1852) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1178..1199) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1527..1546 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1786..1807 /label=EGFP-N /note="EGFP, reverse primer" protein_bind 1866..1884 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1887..1905 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" promoter complement(1907..2110) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 2291..2342 /label=CMVd2 promoter /note="truncated human cytomegalovirus (CMV) immediate early promoter that yields moderately reduced expression relative to the full-length promoter" protein_bind 2344..2362 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2365..2383 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 2410..2433 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2440..2460 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2501..2521 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(2570..2587) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2576..2800 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2933..2952) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3084..3131 /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 3139..4161 /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 4291..4412 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4328..4347) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4382..4401 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4461..4477) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4461..4477) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4474..4496) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4485..4501 /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(4509..4539) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4554..4575 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4692..4709) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4863..5451) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4943..4962) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5622..6482) /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 6245..6264 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6483..6587) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatccgct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc 61 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 121 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 181 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 241 tctccgctcc atcgttcaga tccttatcga tgctatggca gggcctgccg ccccgacgtt 301 ggctgcgagc cctgggcctt cacccgaact tggggggtgg ggtggggaaa aggaagaaac 361 gcgggcgtat tggccccaat ggggtcacgg tggggtatcg acagagtgcc agccctggga 421 ccgaaccccg cgtttatgaa caaacgaccc aacacccgtg cgttttattc tgtcttttta 481 ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca gttagcctcc 541 cccatcagcg aaccgcgggc cctctagatc actagatatg acgcgtgcgg ccgcagatct 601 gggccatttg ttccatgtga gtgctagtaa caggccttgt gtcctgaaat gtactgcgtg 661 gagacgtcag tcagtggcca aaacgtctcc acgcgcagta catttcagca tacagccttc 721 agcaagcctc caggatctgg gccatttgtt ccatgtgagt gctagtaaca ggccttgtgt 781 cctgaaatgt actgcgtgga gacgtcagtc agtggccaaa acgtctccac gcgcagtaca 841 tttcagcata cagccttcag caagcctcca ggatctgggc catttgttcc atgtgagtgc 901 tagtaacagg ccttgtgtcc tgaaatgtac tgcgtggaga cgtcagtcag tggccaaaac 961 gtctccacgc gcagtacatt tcagcataca gccttcagca agcctccagg atcacttaag 1021 actagcgctc caattgcact acctcctctt actacacctt gcgtttcttc tttgggtcga 1081 cctttctctt ctttttaggg tccaccttcc gctttttctt ggggtctgct gcggccttgt 1141 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt 1201 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt 1261 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct 1321 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct 1381 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca 1441 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt 1501 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg 1561 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt 1621 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc 1681 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg 1741 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct 1801 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtcctag 1861 gacgatctct atcactgata gggagatctc tatcactgat agggagagct ctgcttatat 1921 agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac 1981 attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt 2041 ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa 2101 accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt 2161 cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa 2221 tagggggcgt acttggcata tgatacactt gatgtactct agaaaaatgt cgtaacaact 2281 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 2341 ctctccctat cagtgataga gatctcccta tcagtgatag agatcgtcgg atctaagctt 2401 ggtaccatgg attacaagga tgacgacgat aagggatccg aaaacctgta cttccaagga 2461 accggtggtc acctgtacaa tcgatcctgc aggagctagc gagaatttgt attttcaggg 2521 tgatatccag cacagtggca gctgctcgag gtttaaaccc gctgatcagc ctcgactgtg 2581 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2641 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2701 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2761 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 2821 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 2881 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 2941 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 3001 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 3061 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt 3121 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg 3181 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt 3241 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat 3301 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg 3361 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca 3421 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc 3481 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca 3541 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct 3601 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg 3661 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg 3721 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt 3781 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg 3841 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag 3901 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat 3961 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca 4021 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc 4081 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact 4141 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat 4201 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 4261 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4441 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4501 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 4561 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 4621 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 4681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 4741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 4801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 4861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 4921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5281 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 5341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 5521 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 5581 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 5641 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 5701 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 5761 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 5821 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 5881 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 5941 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 6001 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6061 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6121 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6181 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6241 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6301 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6361 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6421 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6481 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 6541 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 6601 aaagtgccac ctgacgtc //