LOCUS       sequence-203369-        5897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with red 
            fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 
            26879144). mRuby3 is brighter than mCherry and has shifted ex and em
            spectra..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5897)
  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 5897)
  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-mRuby3-TEV
FEATURES             Location/Qualifiers
     source          1..5897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    824..842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    845..863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     873..897
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             994..1704
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             1711..1731
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1772..1792
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1853..1870)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1859..2083
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2216..2235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2367..2414
                     /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             2422..3444
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2422..3444
                     /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    3574..3695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3611..3630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3665..3684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3744..3760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3744..3760)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3757..3779)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3768..3784
                     /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(3792..3798,3799..3816,3817..3822))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3837..3858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3975..3992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4146..4734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4226..4245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4905..5696,5697..5765))
                     /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     5528..5547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5766..5870)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga
      841 gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc
      901 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
      961 cggactctgg cgtttaaact taagcttggt accatggtgt ctaagggcga agagctgatc
     1021 aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa
     1081 tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag gatcaaagtc
     1141 atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt catgtatggc
     1201 agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca gtcctttcct
     1261 gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt caccgtcacg
     1321 caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag aggggtaaac
     1381 tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc taatacagag
     1441 atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact gaaagttgat
     1501 ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa gaccgtcggg
     1561 aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat cgaggagagt
     1621 gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag caaccttggt
     1681 ggtggcatgg acgagctgta caagggatcc gaaaacctgt acttccaagg aaccggtggt
     1741 cacctgtaca atcgatcctg caggagctag cgagaatttg tattttcagg gtgatatcca
     1801 gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     1861 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1921 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1981 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2041 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2101 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2161 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2221 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2281 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2341 gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa
     2401 agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     2461 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     2521 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     2581 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     2641 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     2701 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     2761 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     2821 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     2881 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     2941 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     3001 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     3061 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     3121 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     3181 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     3241 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     3301 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     3361 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     3421 actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc
     3481 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     3541 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     3601 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3661 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     3721 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3781 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3841 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3901 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3961 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4021 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4081 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4141 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4201 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4261 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4321 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4381 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4441 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4501 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4561 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4621 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4681 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4741 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4801 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4861 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4921 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4981 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5041 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5101 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5161 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5221 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5281 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5341 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5401 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5461 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5521 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5581 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5641 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5701 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5761 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5821 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5881 cgaaaagtgc cacctga
//
