LOCUS       sequence-200364-        6807 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 6807)
  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 6807)
  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..6807
                     /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        1804..1855
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1857..1875
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1878..1896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1926..1946
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1987..2007
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2014..2016,2017..2019,2020..2733)
                     /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(2759..2776)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2765..2989
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3122..3141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3273..3320
                     /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             3328..4350
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3328..4350
                     /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    4480..4601
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4517..4536)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4571..4590
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4650..4666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4650..4666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4663..4685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4674..4690
                     /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(4698..4704,4705..4722,4723..4728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4743..4764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4881..4898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5052..5640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5132..5151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5811..6602,6603..6671))
                     /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     6434..6453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6672..6776)
                     /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 tctagaaaaa tgtcgtaaca actccgcccc
     1801 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc
     1861 tatcagtgat agagatctcc ctatcagtga tagagatcgt caagcttggt accgagctcg
     1921 gatccgaaaa cctgtacttc caaggaaccg gtggtcacct gtacaatcga tcctgcagga
     1981 gctagcgaga atttgtattt tcagggtgat atcatggtga gcaagggcga ggagctgttc
     2041 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2101 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctcatctgc
     2161 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctcgg ctgcggcctg
     2221 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2281 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2341 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2401 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2461 aacgtctata tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc
     2521 cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc
     2581 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gctaccagtc caagctgagc
     2641 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2701 atcactctcg gcatggacga gctgtacaag taactcgagg tttaaacccg ctgatcagcc
     2761 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2821 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2881 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2941 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3001 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3241 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     3301 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     3361 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3421 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3481 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3541 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     3601 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     3661 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     3721 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     3781 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     3841 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     3901 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     3961 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     4021 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     4081 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     4141 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     4201 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     4261 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4321 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     4381 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4441 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4501 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4561 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4621 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4681 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4741 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4801 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4861 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4921 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4981 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5221 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5461 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5641 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5701 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5761 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5821 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5881 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5941 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6001 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6061 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6121 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6181 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6241 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6301 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6361 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6421 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6481 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6541 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6601 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6661 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6721 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6781 tttccccgaa aagtgccacc tgacgtc
//
