LOCUS Exported 7285 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 C-terminal mCherry.. ACCESSION . VERSION . KEYWORDS pKK-RNAi-nucEGFPmiR-TEV-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7285) 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 7285) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7285 /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 2413..2433 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2474..2494 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2501..3211 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(2646..2664) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2914..2933 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(3237..3254) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3243..3467 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3600..3619) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3751..3798 /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 3806..4828 /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 4958..5079 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4995..5014) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5049..5068 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5128..5144) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5128..5144) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5141..5163) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5152..5168 /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(5176..5206) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5221..5242 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5359..5376) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5530..6118) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5610..5629) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6289..7149) /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 6912..6931 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7150..7254) /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 agatcgtcaa gcttggtacc 2401 gagctcggat ccgaaaacct gtacttccaa ggaaccggtg gtcacctgta caatcgatcc 2461 tgcaggagct agcgagaatt tgtattttca gggtgatatc atggtgagca agggcgagga 2521 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt 2581 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca 2641 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc 2701 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta 2761 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg 2821 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt 2881 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg 2941 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa 3001 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa 3061 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac 3121 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc 3181 caccggcggc atggacgagc tgtacaagta gctcgaggtt taaacccgct gatcagcctc 3241 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 3301 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 3361 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 3421 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga 3481 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc 3541 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3601 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 3661 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 3721 acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct 3781 agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa 3841 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga 3901 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg 3961 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc 4021 gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt gcatctcccg 4081 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca 4141 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt 4201 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc 4261 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc 4321 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca 4381 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc 4441 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt 4501 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca 4561 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca 4621 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg 4681 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag 4741 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc 4801 cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc 4861 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 4921 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 4981 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 5041 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 5101 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 5161 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 5221 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 5281 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 5341 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 5401 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 5461 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 5521 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 5581 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 5641 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5701 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 5761 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5821 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5881 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5941 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 6001 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 6061 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 6121 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 6181 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 6241 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 6301 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 6361 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 6421 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6481 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 6541 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6601 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6661 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6721 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6781 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6841 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6901 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6961 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 7021 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 7081 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 7141 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 7201 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 7261 tccccgaaaa gtgccacctg acgtc //