LOCUS Exported 5894 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: 15558047). mCherry descends from the first truly monomeric red FP mRFP1.. ACCESSION . VERSION . KEYWORDS pKK-mCherry-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5894) 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 5894) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5894 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(39..58) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 230..609 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 610..813 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 764..784 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 815..833 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 836..854 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 864..888 /label=LNCX /note="Human CMV promoter, forward primer" CDS 985..1692 /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(1130..1148) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1398..1417 /label=mCherry-F /note="mCherry, forward primer" CDS 1699..1719 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1760..1780 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(1841..1858) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1847..2071 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2204..2223) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2355..2402 /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 2410..3432 /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 3562..3683 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3599..3618) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3653..3672 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3732..3748) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3732..3748) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3745..3767) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3756..3772 /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(3780..3810) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3825..3846 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3963..3980) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4134..4722) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4214..4233) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4893..5753) /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 5516..5535 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5754..5858) /gene="bla" /label=AmpR promoter ORIGIN 1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca 61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc 121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg 181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 781 cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag agatctccct 841 atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc 901 catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct ccggactctg 961 gcgtttaaac ttaagcttgg taccatggtg agcaagggcg aggaggataa catggccatc 1021 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc 1081 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag 1141 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac 1201 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc 1261 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg 1321 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc 1381 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc 1441 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg 1501 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg 1561 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac 1621 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac 1681 gagctgtaca agggatccga aaacctgtac ttccaaggaa ccggtggtca cctgtacaat 1741 cgatcctgca ggagctagcg agaatttgta ttttcagggt gatatccagc acagtggcgg 1801 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt 1861 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 1921 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 1981 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2041 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc 2101 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2161 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2221 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2281 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 2341 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact 2401 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 2461 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 2521 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 2581 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 2641 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc 2701 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc 2761 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 2821 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 2881 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 2941 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 3001 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 3061 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 3121 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 3181 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 3241 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 3301 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 3361 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 3421 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg 3481 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 3541 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc 3601 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg 3661 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg 3721 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 3781 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 3841 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 3901 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 3961 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4021 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4081 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4141 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4201 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4261 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4321 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4381 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4441 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4501 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4561 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4621 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4681 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4741 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 4801 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 4861 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 4921 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 4981 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 5041 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 5101 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 5161 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5221 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5281 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5341 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5401 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5461 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 5521 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5581 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 5641 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 5701 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 5761 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 5821 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 5881 cctgacgtcg acgg //