LOCUS       sequence-200336-        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(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             990..1700
                     /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             1707..1727
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1768..1788
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1849..1866)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1855..2079
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2212..2231)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2363..2410
                     /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             2418..3440
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2418..3440
                     /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    3570..3691
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3607..3626)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3661..3680
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3740..3756)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3740..3756)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3753..3775)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3764..3780
                     /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(3788..3794,3795..3812,3813..3818))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3833..3854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3971..3988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4142..4730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4222..4241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4901..5692,5693..5761))
                     /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     5524..5543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5762..5866)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtacca tggtgtctaa gggcgaagag ctgatcaagg
     1021 aaaatatgcg tatgaaggtg gtcatggaag gttcggtcaa cggccaccaa ttcaaatgca
     1081 caggtgaagg agaaggcaga ccgtacgagg gagtgcaaac catgaggatc aaagtcatcg
     1141 agggaggacc cctgccattt gcctttgaca ttcttgccac gtcgttcatg tatggcagcc
     1201 gtacctttat caagtacccg gccgacatcc ctgatttctt taaacagtcc tttcctgagg
     1261 gttttacttg ggaaagagtt acgagatacg aagatggtgg agtcgtcacc gtcacgcagg
     1321 acaccagcct tgaggatggc gagctcgtct acaacgtcaa ggtcagaggg gtaaactttc
     1381 cctccaatgg tcccgtgatg cagaagaaga ccaagggttg ggagcctaat acagagatga
     1441 tgtatccagc agatggtggt ctgagaggat acactgacat cgcactgaaa gttgatggtg
     1501 gtggccatct gcactgcaac ttcgtgacaa cttacaggtc aaaaaagacc gtcgggaaca
     1561 tcaagatgcc cggtgtccat gccgttgatc accgcctgga aaggatcgag gagagtgaca
     1621 atgaaaccta cgtagtgcaa agagaagtgg cagttgccaa atacagcaac cttggtggtg
     1681 gcatggacga gctgtacaag ggatccgaaa acctgtactt ccaaggaacc ggtggtcacc
     1741 tgtacaatcg atcctgcagg agctagcgag aatttgtatt ttcagggtga tatccagcac
     1801 agtggcggcc gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     1861 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1921 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1981 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     2041 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     2101 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2161 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2221 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2281 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2341 agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta
     2401 taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     2461 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     2521 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     2581 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     2641 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     2701 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     2761 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     2821 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     2881 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     2941 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     3001 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     3061 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     3121 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     3181 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     3241 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     3301 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     3361 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     3421 gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg
     3481 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3541 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3601 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3661 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3721 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3781 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3841 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3901 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3961 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4021 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4081 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4141 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4201 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4261 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4321 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4381 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4441 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4501 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4561 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4621 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4681 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4741 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4801 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4861 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4921 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4981 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5041 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5101 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5161 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5221 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5281 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5341 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5401 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5461 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5521 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5581 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5641 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5701 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5761 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5821 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5881 aagtgccacc tgacgtc
//
