LOCUS       sequence-203364-        5903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with non-absorbing,
            non-emitting Venus mutant at the N-terminus (cleavable by TEV). 
            Negative control for FRET measurements (PMID: 17040988, 22264545)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5903)
  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 5903)
  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-mAmber-TEV
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     758..778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    809..827
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    830..848
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     858..882
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(979..981,982..984,985..1695)
                     /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(1024..1045)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1285..1304)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1632..1653
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1702..1722
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1763..1783
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1844..1861)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1850..2074
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2207..2226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2358..2405
                     /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             2413..3435
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2413..3435
                     /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    3565..3686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3602..3621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3656..3675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3735..3751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3735..3751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3748..3770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3759..3775
                     /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(3783..3789,3790..3807,3808..3813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3828..3849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3966..3983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4137..4725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4217..4236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4896..5687,5688..5756))
                     /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     5519..5538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5757..5861)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
       61 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
      121 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      181 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      481 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      781 cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt
      841 gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
      901 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac tctggcgttt
      961 aaacttaagc ttggtaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1021 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1081 gagggcgatg ccacctacgg caagctgacc ctgaagctca tctgcaccac cggcaagctg
     1141 cccgtgccct ggcccaccct cgtgaccacc ctcggctgcg gcctgcagtg cttcgcccgc
     1201 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1261 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1321 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1381 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcacc
     1441 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1501 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1561 ctgctgcccg acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag
     1621 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1681 gacgagctgt acaagggatc cgaaaacctg tacttccaag gaaccggtgg tcacctgtac
     1741 aatcgatcct gcaggagcta gcgagaattt gtattttcag ggtgatatcc agcacagtgg
     1801 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     1861 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1921 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     1981 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2041 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2101 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2161 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2221 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2281 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2341 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     2401 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2461 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2521 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2581 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2641 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2701 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2761 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2821 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2881 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     2941 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3001 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3061 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3121 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3181 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3241 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3301 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3361 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3421 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3481 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3541 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3601 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3661 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3721 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3781 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3841 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3901 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3961 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4021 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4081 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4141 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4201 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4261 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4321 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4381 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4441 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4501 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4561 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4621 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4681 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4741 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4801 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4861 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4921 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4981 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5041 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5101 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5161 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5221 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5281 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5341 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5401 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5461 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5521 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5581 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5641 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5701 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5761 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5821 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5881 ccacctgacg tcgacggatc ggg
//
