LOCUS       sequence-203395-        6125 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 6125)
  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 6125)
  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..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..38)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    433..481
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     506..525
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(620..643)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(644..667)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    706..724
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(726..929)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1109..1160
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1162..1180
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1183..1201
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             join(1219..1221,1222..1224,1225..1935)
                     /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(1264..1285)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1872..1893
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1942..1962
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2003..2023
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2072..2089)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2078..2302
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2435..2454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2586..2633
                     /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             2641..3663
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2641..3663
                     /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    3793..3914
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3830..3849)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3884..3903
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3963..3979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3963..3979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3976..3998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3987..4003
                     /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(4011..4017,4018..4035,4036..4041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4056..4077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5124..5915,5916..5984))
                     /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     5747..5766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5985..6089)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ccgctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
       61 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
      121 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
      181 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
      241 gctccatcgt tcagatcctt atcgatgcta tggcagggcc tgccgccccg acgttggctg
      301 cgagccctgg gccttcaccc gaacttgggg ggtggggtgg ggaaaaggaa gaaacgcggg
      361 cgtattggcc ccaatggggt cacggtgggg tatcgacaga gtgccagccc tgggaccgaa
      421 ccccgcgttt atgaacaaac gacccaacac ccgtgcgttt tattctgtct ttttattgcc
      481 gtcatagcgc gggttccttc cggtattgtc tccttccgtg tttcagttag cctcccccat
      541 cagcgaaccg cgggccctta tctacgcgtt tcagagcggc cgcactgatc ttaagggtac
      601 tagcgctgat cggcaattgg ggcccctgga acagaacttc cagcttatcg tcgtcatcct
      661 tgtaatccat aggtcctagg acgatctcta tcactgatag ggagatctct atcactgata
      721 gggagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat
      781 ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc
      841 ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc
      901 ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg
      961 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat
     1021 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactcta
     1081 gaaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     1141 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
     1201 atcgtcaagc ttggtaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1261 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc
     1321 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1381 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc
     1441 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1501 caggagcgca ccatctcttt caaggacgac ggtacctaca agacccgcgc cgaggtgaag
     1561 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1621 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccactacgt ctatatcacg
     1681 gccgacaagc agaagaactg catcaaggct aacttcaaga tccgccacaa cgttgaggac
     1741 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1801 ctgctgcccg acaaccacta cctgagccat cagtccaagc tgagcaaaga ccccaacgag
     1861 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggattac acatggcatg
     1921 gacgagctgt acaagggatc cgaaaacctg tacttccaag gaaccggtgg tcacctgtac
     1981 aatcgatcct gcaggagcta gcgagaattt gtattttcag ggtgatatcc agcacagtgg
     2041 cggccgctcg aggtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2101 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2161 ctttcctaat aaaatgagga aattgtatcg cattgtctga gtaggtgtca ttctattctg
     2221 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2281 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2341 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2401 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2461 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2521 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2581 acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc
     2641 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     2701 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     2761 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     2821 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     2881 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     2941 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     3001 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     3061 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     3121 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     3181 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     3241 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     3301 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     3361 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     3421 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     3481 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     3541 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     3601 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     3661 tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3721 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3781 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3841 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3901 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3961 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4021 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5401 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6121 gacgg
//
