LOCUS       sequence-203399-        6115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Concomitant expression of two proteins. One protein expressed with 
            FLAG, cleavable by 3C protease; second protein expressed with 
            mClover3, cleavable by TEV protease; tags positions: C-termini..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6115)
  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 6115)
  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-ORF1-3C-FLAG_ORF2-TEV-mClover3
FEATURES             Location/Qualifiers
     source          1..6115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..36)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    431..479
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     504..523
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(558..581)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(585..608)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    681..699
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    702..720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(722..925)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1105..1156
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1158..1176
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1179..1197
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1227..1247
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1288..1308
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1315..1317,1318..1320,1321..2034)
                     /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(1360..1381)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1968..1989
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2060..2077)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2066..2290
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2423..2442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2574..2621
                     /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             2629..3651
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2629..3651
                     /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    3781..3902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3818..3837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3872..3891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3951..3967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3951..3967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3964..3986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3975..3991
                     /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(3999..4005,4006..4023,4024..4029))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4044..4065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4182..4199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4353..4941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4433..4452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5112..5903,5904..5972))
                     /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     5735..5754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5973..6077)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgggagatcc gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
       61 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
      121 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
      181 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
      241 tccatcgttc agatccttat cgatgctatg gcagggcctg ccgccccgac gttggctgcg
      301 agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg
      361 tattggcccc aatggggtca cggtggggta tcgacagagt gccagccctg ggaccgaacc
      421 ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt
      481 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatca
      541 gcgaaccgcg ggccttactt atcgtcgtca tccttgtaat ccatgggccc ctggaacaga
      601 acttccagac gcgtttcaga gcggccgcac tgatcttaag ggtactagcg ctgatcggca
      661 attgcgaggt cctaggacga tctctatcac tgatagggag atctctatca ctgataggga
      721 gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
      781 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
      841 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
      901 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
      961 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     1021 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actctagaaa
     1081 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     1141 ctatataagc agagctctcc ctatcagtga tagagatctc cctatcagtg atagagatcg
     1201 tcaagcttgg taccgagctc ggatccgaaa acctgtactt ccaaggaacc ggtggtcacc
     1261 tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga tatcatggtg
     1321 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1381 gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag
     1441 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1501 accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat gaagcagcac
     1561 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcaag
     1621 gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1681 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1741 gagtacaact tcaacagcca ctacgtctat atcacggccg acaagcagaa gaactgcatc
     1801 aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct cgccgaccac
     1861 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1921 agccatcagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1981 gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa gtaactcgag
     2041 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2101 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2161 aatgaggaaa ttgtatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2221 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2281 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2341 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2401 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2461 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2521 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     2581 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     2641 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     2701 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     2761 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     2821 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     2881 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     2941 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     3001 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     3061 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     3121 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     3181 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     3241 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     3301 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     3361 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     3421 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     3481 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     3541 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     3601 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     3661 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3721 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3781 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3841 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3901 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     3961 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     4021 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     4081 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4141 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4201 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4261 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4321 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4381 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4441 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4501 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4561 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4621 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4681 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4741 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4801 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4861 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4921 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4981 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5041 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5101 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5161 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5221 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5281 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5341 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5401 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5461 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5521 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5581 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5641 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5701 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5761 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5821 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5881 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5941 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6001 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6061 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggat
//
