LOCUS Exported 5897 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of your protein of interest in fusion with red fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 26879144). mRuby3 is brighter than mCherry and has shifted ex and em spectra.. ACCESSION . VERSION . KEYWORDS pKK-mRuby3-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5897) 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 5897) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5897 /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 990..1700 /codon_start=1 /product="monomeric red fluorescent protein derived from mRuby2, with improved brightness and photostability (Bajar et al., 2016)" /label=mRuby3 /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV AKYSNLGGGMDELYK" CDS 1707..1727 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1768..1788 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(1849..1866) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1855..2079 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2212..2231) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2363..2410 /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 2418..3440 /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 3570..3691 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3607..3626) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3661..3680 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3740..3756) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3740..3756) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3753..3775) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3764..3780 /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(3788..3818) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3833..3854 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3971..3988) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4142..4730) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4222..4241) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4901..5761) /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 5524..5543 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5762..5866) /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 cttggtacca tggtgtctaa gggcgaagag ctgatcaagg 1021 aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa ttcaaatgca 1081 caggtgaagg agaaggcaga ccgtacgagg gagtgcaaac catgaggatc aaagtcatcg 1141 agggaggacc cctgccattt gcctttgaca ttcttgccac gtcgttcatg tatggcagcc 1201 gtacctttat caagtacccg gccgacatcc ctgatttctt taaacagtcc tttcctgagg 1261 gttttacttg ggaaagagtt acgagatacg aagatggtgg agtcgtcacc gtcacgcagg 1321 acaccagcct tgaggatggc gagctcgtct acaacgtcaa ggtcagaggg gtaaactttc 1381 cctccaatgg tcccgtgatg cagaagaaga ccaagggttg ggagcctaat acagagatga 1441 tgtatccagc agatggtggt ctgagaggat acactgacat cgcactgaaa gttgatggtg 1501 gtggccatct gcactgcaac ttcgtgacaa cttacaggtc aaaaaagacc gtcgggaaca 1561 tcaagatgcc cggtgtccat gccgttgatc accgcctgga aaggatcgag gagagtgaca 1621 atgaaaccta cgtagtgcaa agagaagtgg cagttgccaa atacagcaac cttggtggtg 1681 gcatggacga gctgtacaag ggatccgaaa acctgtactt ccaaggaacc ggtggtcacc 1741 tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga tatccagcac 1801 agtggcggcc gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc 1861 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 1921 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 1981 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 2041 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 2101 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 2161 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 2221 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 2281 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 2341 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta 2401 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga 2461 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 2521 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 2581 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 2641 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac 2701 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 2761 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 2821 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 2881 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 2941 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 3001 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 3061 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 3121 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 3181 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 3241 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 3301 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 3361 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 3421 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg 3481 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg 3541 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca 3601 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3661 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct 3721 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 3781 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 3841 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 3901 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 3961 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 4021 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 4081 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 4141 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 4201 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 4261 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 4321 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 4381 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 4441 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 4501 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 4561 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 4621 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 4681 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 4741 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 4801 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 4861 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 4921 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 4981 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 5041 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 5101 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 5161 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 5221 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 5281 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 5341 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 5401 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 5461 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 5521 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 5581 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 5641 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 5701 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 5761 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5821 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 5881 aagtgccacc tgacgtc //