LOCUS       Exported                6168 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of your protein of interest in fusion with BirA-FLAG at 
            the C-terminus (cleavable by TEV). Search for protein partners using
            the BioID (PMID: 15707685, 22412018)..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-TEV-BirA-FLAG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6168)
  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 6168)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6168
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(42..61)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        233..612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     767..787
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    818..836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    839..857
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     867..891
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1000..1020
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1061..1081
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1088..2050
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2054..2077
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2118..2135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2124..2348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2481..2500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2632..2679
                     /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             2687..3709
                     /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    3839..3960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3876..3895)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3930..3949
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4009..4025)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4009..4025)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4022..4044)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4033..4049
                     /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(4057..4087)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4102..4123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4240..4257)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4411..4999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4491..4510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5170..6030)
                     /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     5793..5812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6031..6135)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
       61 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
      121 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      181 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      241 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      301 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      421 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      781 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
      841 cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga
      901 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggact
      961 ctggcgttta aacttaagct tggtaccgag ctcggatccg aaaacctgta cttccaagga
     1021 accggtggtc acctgtacaa tcgatcctgc aggagctagc gagaatttgt attttcaggg
     1081 tgatatcatg aaggataaca ccgtgccact gaaattgatt gccctgttag cgaacggtga
     1141 atttcactct ggcgagcagt tgggtgaaac gctgggaatg agccgggcgg ctattaataa
     1201 acacattcag acactgcgtg actggggcgt tgatgtcttt accgttccgg gtaaaggata
     1261 cagcctgcct gagcctatcc agttacttaa tgctaaacag atattgggtc agctggatgg
     1321 cggtagtgta gccgtgctgc cagtgattga ctccacgaat cagtaccttc ttgatcgtat
     1381 cggagagctt aaatcgggcg atgcttgcat tgcagaatac cagcaggctg gccgtggtcg
     1441 ccggggtcgg aaatggtttt cgccttttgg cgcaaactta tatttgtcga tgttctggcg
     1501 tctggaacaa ggcccggcgg cggcgattgg tttaagtctg gttatcggta tcgtgatggc
     1561 ggaagtatta cgcaagctgg gtgcagataa agttcgtgtt aaatggccta atgacctcta
     1621 tctgcaggat cgcaagctgg caggcattct ggtggagctg actggcaaaa ctggcgatgc
     1681 ggcgcaaata gtcattggag ccgggatcaa catggcaatg cgccgtgttg aagagagtgt
     1741 cgttaatcag gggtggatca cgctgcagga agcggggatc aatctcgatc gtaatacgtt
     1801 ggcggccatg ctaatacgtg aattacgtgc tgcgttggaa ctcttcgaac aagaaggatt
     1861 ggcaccttat ctgtcgcgct gggaaaagct ggataatttt attaatcgcc cagtgaaact
     1921 tatcattggt gataaagaaa tatttggcat ttcacgcgga atagacaaac agggggcttt
     1981 attacttgag caggatggaa taataaaacc ctggatgggc ggtgaaatat ccctgcgtag
     2041 tgcagaaaaa atcgattaca aggatgacga cgataagtag ctcgagtcta gagggcccgt
     2101 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2161 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2221 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2281 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2341 ctctatggct tctaaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2401 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2461 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2521 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2581 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     2641 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     2701 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     2761 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     2821 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     2881 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     2941 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     3001 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     3061 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     3121 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     3181 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     3241 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     3301 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     3361 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     3421 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     3481 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     3541 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     3601 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     3661 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     3721 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3781 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3841 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3901 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3961 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4021 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4081 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4141 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4201 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4261 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4321 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4381 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4441 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4501 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4561 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4621 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4681 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4741 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4801 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4861 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4921 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4981 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5041 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5101 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5161 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5221 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5281 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5341 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5401 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5461 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5521 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5581 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5641 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5701 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5761 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5821 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5881 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5941 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6001 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6061 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6121 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcga
//
