LOCUS Exported 5895 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of your protein of interest in fusion with cyan fluorescent protein at the C-terminus (cleavable by TEV). Useful for FRET experiments (PMID: 14990965, 17040988, 22264545). . ACCESSION . VERSION . KEYWORDS pKK-TEV-mCerulean SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5895) 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 5895) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5895 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(36..55) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 227..606 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 607..810 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 761..781 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 812..830 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 833..851 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 861..885 /label=LNCX /note="Human CMV promoter, forward primer" CDS 994..1014 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1055..1075 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1082..1801 /codon_start=1 /product="enhanced monomeric variant of CFP (Rizzo et al., 2004)" /label=mCerulean /note=" mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1127..1148) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1388..1407) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1735..1756 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1839..1856) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1845..2069 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2202..2221) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2353..2400 /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 2408..3430 /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 3560..3681 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3597..3616) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3651..3670 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3730..3746) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3730..3746) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3743..3765) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3754..3770 /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(3778..3808) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3823..3844 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3961..3978) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4132..4720) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4212..4231) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4891..5751) /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 5514..5533 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5752..5856) /gene="bla" /label=AmpR promoter ORIGIN 1 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag 61 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa 121 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt 181 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg 241 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 301 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 361 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 421 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 481 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 541 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 601 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 661 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 721 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt 781 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc 841 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat 901 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctggcg 961 tttaaactta agcttggtac cgagctcgga tccgaaaacc tgtacttcca aggaaccggt 1021 ggtcacctgt acaatcgatc ctgcaggagc tagcgagaat ttgtattttc agggtgatat 1081 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 1141 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 1201 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac 1261 cctcgtgacc accctgacct ggggcgtgca gtgcttcgcc cgctaccccg accacatgaa 1321 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 1381 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 1441 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 1501 caagctggag tacaacgcca tcagcgacaa cgtctatatc accgccgaca agcagaagaa 1561 cggcatcaag gccaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc 1621 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 1681 ctacctgagc acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt 1741 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta 1801 actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 1861 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 1921 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 1981 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 2041 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 2101 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 2161 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 2221 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 2281 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 2341 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc 2401 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt 2461 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt 2521 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa 2581 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga 2641 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac 2701 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat 2761 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca 2821 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt 2881 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga 2941 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt 3001 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga 3061 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt 3121 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc 3181 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt 3241 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc 3301 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga 3361 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc 3421 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg 3481 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct 3541 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa 3601 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 3661 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc 3721 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 3781 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 3841 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 3901 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 3961 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4021 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4081 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4141 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 4201 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 4261 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 4321 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 4381 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 4441 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 4501 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 4561 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 4621 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 4681 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4741 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 4801 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 4861 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 4921 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 4981 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5041 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5101 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 5161 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 5221 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 5281 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 5341 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 5401 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 5461 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 5521 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 5581 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 5641 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 5701 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 5761 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 5821 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 5881 tgacgtcgac ggatc //