LOCUS       sequence-203337-        6182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with FLAG-BirA. 
            FLAG-tagged at the N-terminus (cleavable by TEV). Search for protein
            partners using the BioID (PMID: 15707685, 22412018)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  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 6182)
  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-FLAG-BirA-TEV
FEATURES             Location/Qualifiers
     source          1..6182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             985..1008
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1015..1977
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1015..1977
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1984..2004
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2045..2065
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2126..2143)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2132..2356
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2489..2508)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2640..2687
                     /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             2695..3717
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFCESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3847..3968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3884..3903)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3938..3957
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4017..4033)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4017..4033)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4030..4052)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4041..4057
                     /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(4065..4071,4072..4089,4090..4095))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4110..4131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4248..4265)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4419..5007)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4499..4518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5178..5969,5970..6038))
                     /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     5801..5820
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6039..6143)
                     /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 catggattac aaggatgacg acgataaggg atccatgaag
     1021 gataacaccg tgccactgaa attgattgcc ctgttagcga acggtgaatt tcactctggc
     1081 gagcagttgg gtgaaacgct gggaatgagc cgggcggcta ttaataaaca cattcagaca
     1141 ctgcgtgact ggggcgttga tgtctttacc gttccgggta aaggatacag cctgcctgag
     1201 cctatccagt tacttaatgc taaacagata ttgggtcagc tggatggcgg tagtgtagcc
     1261 gtgctgccag tgattgactc cacgaatcag taccttcttg atcgtatcgg agagcttaaa
     1321 tcgggcgatg cttgcattgc agaataccag caggctggcc gtggtcgccg gggtcggaaa
     1381 tggttttcgc cttttggcgc aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc
     1441 ccggcggcgg cgattggttt aagtctggtt atcggtatcg tgatggcgga agtattacgc
     1501 aagctgggtg cagataaagt tcgtgttaaa tggcctaatg acctctatct gcaggatcgc
     1561 aagctggcag gcattctggt ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc
     1621 attggagccg ggatcaacat ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg
     1681 tggatcacgc tgcaggaagc ggggatcaat ctcgatcgta atacgttggc ggccatgcta
     1741 atacgtgaat tacgtgctgc gttggaactc ttcgaacaag aaggattggc accttatctg
     1801 tcgcgctggg aaaagctgga taattttatt aatcgcccag tgaaacttat cattggtgat
     1861 aaagaaatat ttggcatttc acgcggaata gacaaacagg gggctttatt acttgagcag
     1921 gatggaataa taaaaccctg gatgggcggt gaaatatccc tgcgtagtgc agaaaaagga
     1981 tccgaaaacc tgtacttcca aggaaccggt ggtcacctgt acaatcgatc ctgcaggagc
     2041 tagcgagaat ttgtattttc agggtgatat ccagcacagt ggcggccgct cgagtctaga
     2101 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2161 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2221 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2281 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2341 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     2401 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2461 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2521 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2581 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag
     2641 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag
     2701 cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc
     2761 gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
     2821 cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt
     2881 tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc
     2941 tgcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg
     3001 cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct
     3061 gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
     3121 tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa
     3181 actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt
     3241 tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat
     3301 gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg
     3361 gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
     3421 cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg
     3481 gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc
     3541 gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact
     3601 gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa
     3661 gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac
     3721 gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3781 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3841 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3901 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3961 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4021 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4081 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4141 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4201 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4261 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4321 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4381 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4441 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4501 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4561 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4621 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4681 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4741 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4801 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4861 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4921 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5161 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5221 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5281 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5341 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5401 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5461 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5521 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5581 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5641 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5701 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5761 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5821 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5881 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5941 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6001 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6061 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6121 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     6181 tc
//
