LOCUS Exported 6125 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Concomitant expression of two proteins. One protein expressed with FLAG, cleavable by 3C or enterokinase; second protein expressed with mClover3, cleavable by TEV protease; tags positions: N-termini.. ACCESSION . VERSION . KEYWORDS pKK-BI16-FLAG-3C-ORF1_mClover3-TEV-ORF2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6125) 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 6125) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6125 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(18..38) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" polyA_signal 433..481 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind 506..525 /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" CDS complement(620..643) /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS complement(644..667) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" protein_bind 685..703 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 706..724 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" promoter complement(726..929) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 1109..1160 /label=CMVd2 promoter /note="truncated human cytomegalovirus (CMV) immediate early promoter that yields moderately reduced expression relative to the full-length promoter" protein_bind 1162..1180 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 1183..1201 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 1219..1935 /codon_start=1 /product="bright green-yellow fluorescent protein derived from mClover2, with improved photostability (Bajar et al., 2003)" /label=mClover3 /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" primer_bind complement(1264..1285) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 1872..1893 /label=EGFP-C /note="EGFP, forward primer" CDS 1942..1962 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 2003..2023 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" primer_bind complement(2072..2089) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2078..2302 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(2435..2454) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 2586..2633 /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 2641..3663 /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 3793..3914 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3830..3849) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3884..3903 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3963..3979) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3963..3979) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3976..3998) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3987..4003 /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(4011..4041) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4056..4077 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4194..4211) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4365..4953) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4445..4464) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5124..5984) /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 5747..5766 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5985..6089) /gene="bla" /label=AmpR promoter ORIGIN 1 atcgggagat ccgctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 61 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 121 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 181 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 241 gctccatcgt tcagatcctt atcgatgcta tggcagggcc tgccgccccg acgttggctg 301 cgagccctgg gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg 361 cgtattggcc ccaatggggt cacggtgggg tatcgacaga gtgccagccc tgggaccgaa 421 ccccgcgttt atgaacaaac gacccaacac ccgtgcgttt tattctgtct ttttattgcc 481 gtcatagcgc gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccat 541 cagcgaaccg cgggccctta tctacgcgtt tcagagcggc cgcactgatc ttaagggtac 601 tagcgctgat cggcaattgg ggcccctgga acagaacttc cagcttatcg tcgtcatcct 661 tgtaatccat aggtcctagg acgatctcta tcactgatag ggagatctct atcactgata 721 gggagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat 781 ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc 841 ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc 901 ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg 961 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat 1021 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactcta 1081 gaaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 1141 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag 1201 atcgtcaagc ttggtaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 1261 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc 1321 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 1381 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc 1441 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 1501 caggagcgca ccatctcttt caaggacgac ggtacctaca agacccgcgc cgaggtgaag 1561 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1621 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccactacgt ctatatcacg 1681 gccgacaagc agaagaactg catcaaggct aacttcaaga tccgccacaa cgttgaggac 1741 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1801 ctgctgcccg acaaccacta cctgagccat cagtccaagc tgagcaaaga ccccaacgag 1861 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggattac acatggcatg 1921 gacgagctgt acaagggatc cgaaaacctg tacttccaag gaaccggtgg tcacctgtac 1981 aatcgatcct gcaggagcta gcgagaattt gtattttcag ggtgatatcc agcacagtgg 2041 cggccgctcg aggtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca 2101 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 2161 ctttcctaat aaaatgagga aattgtatcg cattgtctga gtaggtgtca ttctattctg 2221 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 2281 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 2341 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 2401 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 2461 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 2521 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 2581 acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc 2641 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc 2701 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta 2761 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt 2821 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg 2881 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa 2941 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg 3001 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc 3061 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac 3121 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg 3181 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc 3241 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg 3301 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt 3361 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg 3421 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc 3481 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc 3541 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt 3601 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa 3661 tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 3721 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 3781 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 3841 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 3901 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca 3961 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4021 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4801 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5401 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6121 gacgg //