LOCUS Exported 7284 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 7284) 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 7284) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7284 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(28..48) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" polyA_signal 443..491 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind 516..535 /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" ncRNA complement(607..648) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(712..738) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(745..786) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(850..876) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(883..924) /label=3' miR-155 /note="sequence downstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" ncRNA complement(988..1014) /label=5' miR-155 /note="sequence upstream of the precursor of mouse miR-155 microRNA (Uva et al., 2013)" /ncRNA_class="miRNA" CDS complement(1060..1080) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1084..1104) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1108..1128) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(1141..1857) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(1141..1857) /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(1183..1204) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1532..1551 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1791..1812 /label=EGFP-N /note="EGFP, reverse primer" protein_bind 1871..1889 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1892..1910 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" promoter complement(1912..2115) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 2295..2346 /label=CMVd2 promoter /note="truncated human cytomegalovirus (CMV) immediate early promoter that yields moderately reduced expression relative to the full-length promoter" protein_bind 2348..2366 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2369..2387 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 2417..2437 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2478..2498 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2505..3215 /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(2650..2668) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2918..2937 /label=mCherry-F /note="mCherry, forward primer" primer_bind complement(3241..3258) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3247..3471 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3604..3623) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3755..3802 /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 3810..4832 /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 4962..5083 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4999..5018) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5053..5072 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5132..5148) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5132..5148) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5145..5167) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5156..5172 /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(5180..5210) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5225..5246 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5363..5380) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5534..6122) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5614..5633) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6293..7153) /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 6916..6935 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7154..7258) /gene="bla" /label=AmpR promoter ORIGIN 1 acgtcgacgg atcgggagat ccgctgtgga atgtgtgtca gttagggtgt ggaaagtccc 61 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 121 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 181 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg 241 cccattctcc gctccatcgt tcagatcctt atcgatgcta tggcagggcc tgccgccccg 301 acgttggctg cgagccctgg gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa 361 gaaacgcggg cgtattggcc ccaatggggt cacggtgggg tatcgacaga gtgccagccc 421 tgggaccgaa ccccgcgttt atgaacaaac gacccaacac ccgtgcgttt tattctgtct 481 ttttattgcc gtcatagcgc gggttccttc cggtattgtc tccttccgtg tttcagttag 541 cctcccccat cagcgaaccg cgggccctct agatcactag atatgacgcg tgcggccgca 601 gatctgggcc atttgttcca tgtgagtgct agtaacaggc cttgtgtcct gaaatgtact 661 gcgtggagac gtcagtcagt ggccaaaacg tctccacgcg cagtacattt cagcatacag 721 ccttcagcaa gcctccagga tctgggccat ttgttccatg tgagtgctag taacaggcct 781 tgtgtcctga aatgtactgc gtggagacgt cagtcagtgg ccaaaacgtc tccacgcgca 841 gtacatttca gcatacagcc ttcagcaagc ctccaggatc tgggccattt gttccatgtg 901 agtgctagta acaggccttg tgtcctgaaa tgtactgcgt ggagacgtca gtcagtggcc 961 aaaacgtctc cacgcgcagt acatttcagc atacagcctt cagcaagcct ccaggatcac 1021 ttaagactag cgctccaatt gcactacctc ctcttactac accttgcgtt tcttctttgg 1081 gtcgaccttt ctcttctttt tagggtccac cttccgcttt ttcttggggt ctgctgcggc 1141 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac 1201 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg 1261 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc 1321 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt 1381 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg 1441 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag 1501 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa 1561 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt 1621 catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt 1681 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta 1741 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc 1801 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt 1861 cctaggacga tctctatcac tgatagggag atctctatca ctgataggga gagctctgct 1921 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt 1981 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat 2041 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg 2101 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg 2161 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac 2221 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actctagaaa atgtcgtaac 2281 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 2341 agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg tcaagcttgg 2401 taccgagctc ggatccgaaa acctgtactt ccaaggaacc ggtggtcacc tgtacaatcg 2461 atcctgcagg agctagcgag aatttgtatt ttcagggtga tatcatggtg agcaagggcg 2521 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct 2581 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca 2641 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc 2701 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg 2761 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg 2821 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca 2881 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca 2941 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga 3001 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct 3061 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca 3121 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc 3181 actccaccgg cggcatggac gagctgtaca agtagctcga ggtttaaacc cgctgatcag 3241 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 3301 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgtatcgc 3361 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 3421 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 3481 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa 3541 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 3601 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3661 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3721 aaaaacttga ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt 3781 ctctagaaag tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg 3841 agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 3901 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 3961 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 4021 tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct 4081 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 4141 tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg 4201 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 4261 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 4321 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 4381 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 4441 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 4501 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 4561 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg 4621 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 4681 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 4741 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 4801 gccccagcac tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg 4861 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc 4921 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt 4981 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 5041 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt 5101 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 5161 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 5221 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 5281 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 5341 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 5401 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 5461 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 5521 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 5581 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 5641 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 5701 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 5761 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 5821 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 5881 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5941 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 6001 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 6061 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 6121 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 6181 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 6241 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 6301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 6361 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 6421 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 6481 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 6541 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 6601 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 6661 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 6721 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 6781 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 6841 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 6901 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 6961 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 7021 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 7081 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 7141 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 7201 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 7261 catttccccg aaaagtgcca cctg //