LOCUS       sequence-203373-        5250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with 3xFLAG at the 
            C-terminus. The tag is cleavable by TEV protease..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5250)
  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 5250)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2018-03-28"; volume: "13(3):e0194887. doi"; pages: " 
            10.1371/journal.pone.0194887. eCollection 2018"
COMMENT     Sequence Label: pKK-TEV-3XFLAG
FEATURES             Location/Qualifiers
     source          1..5250
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    823..841
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    844..862
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     872..896
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1005..1025
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1066..1086
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1111..1176
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1205..1222)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1211..1435
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1568..1587)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1719..1766
                     /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             1774..2796
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1774..2796
                     /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    2926..3047
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2963..2982)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3017..3036
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3096..3112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3096..3112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3109..3131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3120..3136
                     /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(join(3144..3150,3151..3168,3169..3174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3189..3210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3327..3344)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3498..4086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3578..3597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4257..5048,5049..5117))
                     /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     4880..4899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5118..5222)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag
      841 atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct
      901 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
      961 ggactctggc gtttaaactt aagcttggta ccgagctcgg atccgaaaac ctgtacttcc
     1021 aaggaaccgg tggtcacctg tacaatcgat cctgcaggag ctagcgagaa tttgtatttt
     1081 cagggtgata tccagcacag tggcggccgc gactacaaag accatgacgg tgattataaa
     1141 gatcatgaca tcgattacaa ggatgacgat gacaagtaag ggcccgttta aacccgctga
     1201 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1261 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1321 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1381 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     1441 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     1501 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1561 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     1621 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     1681 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     1741 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     1801 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     1861 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     1921 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     1981 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2041 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2101 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2161 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2221 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2281 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2341 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     2401 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     2461 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     2521 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     2581 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     2641 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     2701 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     2761 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     2821 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     2881 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     2941 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3001 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3061 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3121 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3181 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3241 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3301 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3361 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3421 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3481 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5221 cgcacatttc cccgaaaagt gccacctgac
//
