LOCUS       sequence-200359-        6126 bp    DNA     circular SYN 15-DEC-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  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 6126)
  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-BI16-FLAG-3C-ORF1_mClover3-TEV-ORF2
FEATURES             Location/Qualifiers
     source          1..6126
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..43)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    438..486
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     511..530
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(625..648)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(649..672)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    690..708
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    711..729
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(731..934)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1115..1166
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1168..1186
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1189..1207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(1225..1227,1228..1230,1231..1941)
                     /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(1270..1291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1878..1899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1948..1968
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2009..2029
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2078..2095)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2084..2308
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2441..2460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2592..2639
                     /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             2647..3669
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2647..3669
                     /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    3799..3920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3836..3855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3890..3909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3969..3985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3969..3985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3982..4004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3993..4009
                     /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(4017..4023,4024..4041,4042..4047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4062..4083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4200..4217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4371..4959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4451..4470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5130..5921,5922..5990))
                     /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     5753..5772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5991..6095)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatccgct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
       61 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
      121 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      181 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
      241 tctccgctcc atcgttcaga tccttatcga tgctatggca gggcctgccg ccccgacgtt
      301 ggctgcgagc cctgggcctt cacccgaact tggggggtgg ggtggggaaa aggaagaaac
      361 gcgggcgtat tggccccaat ggggtcacgg tggggtatcg acagagtgcc agccctggga
      421 ccgaaccccg cgtttatgaa caaacgaccc aacacccgtg cgttttattc tgtcttttta
      481 ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca gttagcctcc
      541 cccatcagcg aaccgcgggc ccttatctac gcgtttcaga gcggccgcac tgatcttaag
      601 ggtactagcg ctgatcggca attggggccc ctggaacaga acttccagct tatcgtcgtc
      661 atccttgtaa tccataggtc ctaggacgat ctctatcact gatagggaga tctctatcac
      721 tgatagggag agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg
      781 tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca
      841 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc
      901 cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt
      961 agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg
     1021 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta
     1081 ctctagaaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     1141 ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg
     1201 atagagatcg tcaagcttgg taccatggtg agcaagggcg aggagctgtt caccggggtg
     1261 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc
     1321 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc
     1381 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc
     1441 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1501 tacgtccagg agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag
     1561 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1621 gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca ctacgtctat
     1681 atcacggccg acaagcagaa gaactgcatc aaggctaact tcaagatccg ccacaacgtt
     1741 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1801 cccgtgctgc tgcccgacaa ccactacctg agccatcagt ccaagctgag caaagacccc
     1861 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gattacacat
     1921 ggcatggacg agctgtacaa gggatccgaa aacctgtact tccaaggaac cggtggtcac
     1981 ctgtacaatc gatcctgcag gagctagcga gaatttgtat tttcagggtg atatccagca
     2041 cagtggcggc cgctcgaggt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2101 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2161 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2221 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2281 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2341 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2401 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2461 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2521 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2581 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     2641 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     2701 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     2761 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     2821 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     2881 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     2941 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     3001 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     3061 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     3121 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     3181 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     3241 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     3301 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     3361 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     3421 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     3481 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     3541 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     3601 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     3661 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3721 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3781 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3841 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3901 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3961 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4021 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4201 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4801 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4921 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5041 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5101 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5161 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5221 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5281 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5341 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5401 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5461 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5521 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5581 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5641 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5701 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5761 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5821 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5881 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5941 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6001 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6061 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6121 gacgtc
//
