LOCUS Exported 5885 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of your protein of interest in fusion with blue fluorescent protein at the N-terminus (cleavable by TEV) (PMID: 18940671, 25378625). Useful for multi-channel live cell imaging.. ACCESSION . VERSION . KEYWORDS pKK-mTagBFP-TEV SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5885) 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 5885) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5885 /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..1688 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" CDS 1695..1715 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1756..1776 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(1837..1854) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1843..2067 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2200..2219) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2351..2398 /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 2406..3428 /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 3558..3679 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3595..3614) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3649..3668 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3728..3744) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3728..3744) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3741..3763) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3752..3768 /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(3776..3806) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3821..3842 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3959..3976) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4130..4718) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4210..4229) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4889..5749) /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 5512..5531 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5750..5854) /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 ctctggcgtt taaacttaag cttggtacca tgagcgagct gattaaggag aacatgcaca 1021 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg 1081 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc 1141 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca 1201 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg 1261 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc 1321 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc 1381 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg 1441 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga 1501 tcgcaaacat caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg 1561 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg 1621 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca 1681 agcttaatgg atccgaaaac ctgtacttcc aaggaaccgg tggtcacctg tacaatcgat 1741 cctgcaggag ctagcgagaa tttgtatttt cagggtgata tccagcacag tggcggccgc 1801 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc 1861 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 1921 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 1981 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 2041 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta 2101 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 2161 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 2221 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 2281 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 2341 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct 2401 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg 2461 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg 2521 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag 2581 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca 2641 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt 2701 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg 2761 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag 2821 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt 2881 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg 2941 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg 3001 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg 3061 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg 3121 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc 3181 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg 3241 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg 3301 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg 3361 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa 3421 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 3481 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 3541 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 3601 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 3661 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt 3721 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 3781 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 3841 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 3901 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 3961 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 4021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 4081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4561 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4861 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4921 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4981 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 5041 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 5101 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 5161 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 5221 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 5281 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 5341 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5401 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5461 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5521 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5581 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5641 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5701 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5761 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5821 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5881 acgtc //