LOCUS       sequence-207907-        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Concomitant expression of two proteins. One protein expressed with 
            Cerulean, cleavable by 3C; second protein expressed with Amber, 
            cleavable by TEV. Control for protein interaction analysis by FRET..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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-FRET-ORF1-3C-Cerulean_ORF2-TEV-Amber
FEATURES             Location/Qualifiers
     source          1..6806
                     /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(join(563..1276,1277..1279,1280..1282))
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(1283..1306)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1379..1397
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1400..1418
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1420..1623)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1803..1854
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1856..1874
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1877..1895
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1925..1945
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1986..2006
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2013..2015,2016..2018,2019..2732)
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002; Kremers et al., 2006)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGCGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2758..2775)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2764..2988
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3121..3140)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3272..3319
                     /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             3327..4349
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3327..4349
                     /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    4479..4600
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4516..4535)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4570..4589
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4649..4665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4649..4665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4662..4684)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4673..4689
                     /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(4697..4703,4704..4721,4722..4727))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4742..4763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4880..4897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5051..5639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5131..5150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5810..6601,6602..6670))
                     /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     6433..6452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6671..6775)
                     /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 ccttacttgt acagctcgtc catgccgaga gtgatcccgg
      601 cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca
      661 gcttggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg
      721 gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga
      781 tcttgaagtt ggccttgatg ccgttcttct gcttgtcggc ggtgatatag acgttgtcgc
      841 tgatggcgtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
      901 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
      961 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg
     1021 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcgggcgaag cactgcacgc
     1081 cccaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
     1141 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga
     1201 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
     1261 gctcctcgcc cttgctcacc atgggcccct ggaacagaac ttccagacgc gtttcagagc
     1321 ggccgcactg atcttaaggg tactagcgct gatcggcaat tgcgaggtcc taggacgatc
     1381 tctatcactg atagggagat ctctatcact gatagggaga gctctgctta tatagacctc
     1441 ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg
     1501 aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact
     1561 tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat
     1621 caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa
     1681 ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg
     1741 cgtacttggc atatgataca cttgatgtac tctagaaaat gtcgtaacaa ctccgcccca
     1801 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct
     1861 atcagtgata gagatctccc tatcagtgat agagatcgtc aagcttggta ccgagctcgg
     1921 atccgaaaac ctgtacttcc aaggaaccgg tggtcacctg tacaatcgat cctgcaggag
     1981 ctagcgagaa tttgtatttt cagggtgata tcatggtgag caagggcgag gagctgttca
     2041 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2101 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctcatctgca
     2161 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctcggc tgcggcctgc
     2221 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2281 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2341 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2401 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2461 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     2521 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2581 gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc aagctgagca
     2641 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2701 tcactctcgg catggacgag ctgtacaagt aactcgaggt ttaaacccgc tgatcagcct
     2761 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2821 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gtatcgcatt
     2881 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2941 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3001 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     3061 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     3121 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3181 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3241 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     3301 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     3361 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3421 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3481 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3541 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3601 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     3661 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     3721 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     3781 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     3841 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     3901 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     3961 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4021 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4081 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4141 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4201 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4261 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4321 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     4381 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4441 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4501 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4561 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4621 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4681 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4741 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4801 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4861 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4921 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4981 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5041 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5101 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5161 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5221 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5281 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5341 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5401 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5461 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5521 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5581 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5641 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5701 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5761 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5821 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5881 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5941 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6001 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6061 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6121 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6181 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6241 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6301 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6361 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6421 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6481 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6541 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6601 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6661 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6721 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6781 ttccccgaaa agtgccacct gacgtc
//
