LOCUS       sequence-203372-        5202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with FLAG 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 5202)
  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 5202)
  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-FLAG
FEATURES             Location/Qualifiers
     source          1..5202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..59)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        231..610
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..814
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     765..785
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    816..834
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    837..855
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     865..889
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             998..1018
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1059..1079
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1086..1109
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1150..1167)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1156..1380
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1513..1532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    1664..1711
                     /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             1719..2741
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1719..2741
                     /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    2871..2992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2908..2927)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2962..2981
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3041..3057)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3041..3057)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3054..3076)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3065..3081
                     /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(3089..3095,3096..3113,3114..3119))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3134..3155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3272..3289)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3443..4031)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3523..3542)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4202..4993,4994..5062))
                     /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     4825..4844
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5063..5167)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
       61 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
      121 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      181 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      241 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      301 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      361 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      421 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      481 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      541 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      601 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      661 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      721 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      781 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
      841 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
      901 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
      961 ggcgtttaaa cttaagcttg gtaccgagct cggatccgaa aacctgtact tccaaggaac
     1021 cggtggtcac ctgtacaatc gatcctgcag gagctagcga gaatttgtat tttcagggtg
     1081 atatcgatta caaggatgac gacgataagt agctcgagtc tagagggccc gtttaaaccc
     1141 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1201 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1261 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1321 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     1381 cttctaaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     1441 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1501 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1561 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1621 tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa
     1681 gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga
     1741 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct
     1801 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc
     1861 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat
     1921 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct
     1981 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc
     2041 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc
     2101 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg
     2161 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca
     2221 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc
     2281 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg
     2341 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg
     2401 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact
     2461 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca
     2521 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg
     2581 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa
     2641 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg
     2701 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg
     2761 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     2821 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     2881 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2941 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     3001 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     3061 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3121 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3181 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3241 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     3301 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     3361 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     3421 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3481 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3541 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3601 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3661 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3721 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3781 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3841 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3901 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3961 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4021 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4081 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4141 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4201 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4261 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4321 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4381 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4441 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4501 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4561 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4621 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4681 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4741 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4801 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4861 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4921 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4981 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5041 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     5101 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     5161 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cg
//
