LOCUS       sequence-203396-        6799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Concomitant expression of two proteins. One protein expressed with 
            mRuby3, cleavable by 3C; second protein expressed with mClover3, 
            cleavable by TEV protease; tags positions: C-termini..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6799)
  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 6799)
  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-BI16-ORF1-3C-mRuby3_ORF2-TEV-mClover3
FEATURES             Location/Qualifiers
     source          1..6799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..35)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    430..478
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     503..522
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(557..1267)
                     /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             complement(1268..1291)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1364..1382
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1385..1403
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1405..1608)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1788..1839
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1841..1859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1862..1880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1910..1930
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1971..1991
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1998..2000,2001..2003,2004..2717)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2043..2064)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2651..2672
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2743..2760)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2749..2973
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3106..3125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3257..3304
                     /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             3312..4334
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3312..4334
                     /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    4464..4585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4501..4520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4555..4574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4634..4650)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4634..4650)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4647..4669)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4658..4674
                     /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(4682..4688,4689..4706,4707..4712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4727..4748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4865..4882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5036..5624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5116..5135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5795..6586,6587..6655))
                     /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     6418..6437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6656..6760)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gggagatccg ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
       61 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
      121 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
      181 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgct
      241 ccatcgttca gatccttatc gatgctatgg cagggcctgc cgccccgacg ttggctgcga
      301 gccctgggcc ttcacccgaa cttggggggt ggggtgggga aaaggaagaa acgcgggcgt
      361 attggcccca atggggtcac ggtggggtat cgacagagtg ccagccctgg gaccgaaccc
      421 cgcgtttatg aacaaacgac ccaacacccg tgcgttttat tctgtctttt tattgccgtc
      481 atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct cccccatcag
      541 cgaaccgcgg gccttacttg tacagctcgt ccatgccacc accaaggttg ctgtatttgg
      601 caactgccac ttctctttgc actacgtagg tttcattgtc actctcctcg atcctttcca
      661 ggcggtgatc aacggcatgg acaccgggca tcttgatgtt cccgacggtc ttttttgacc
      721 tgtaagttgt cacgaagttg cagtgcagat ggccaccacc atcaactttc agtgcgatgt
      781 cagtgtatcc tctcagacca ccatctgctg gatacatcat ctctgtatta ggctcccaac
      841 ccttggtctt cttctgcatc acgggaccat tggagggaaa gtttacccct ctgaccttga
      901 cgttgtagac gagctcgcca tcctcaaggc tggtgtcctg cgtgacggtg acgactccac
      961 catcttcgta tctcgtaact ctttcccaag taaaaccctc aggaaaggac tgtttaaaga
     1021 aatcagggat gtcggccggg tacttgataa aggtacggct gccatacatg aacgacgtgg
     1081 caagaatgtc aaaggcaaat ggcaggggtc ctccctcgat gactttgatc ctcatggttt
     1141 gcactccctc gtacggtctg ccttctcctt cacctgtgca tttgaattgg tggccgttga
     1201 ccgaaccttc catgaccacc ttcatacgca tattttcctt gatcagctct tcgcccttag
     1261 acaccatggg cccctggaac agaacttcca gacgcgtttc agagcggccg cactgatctt
     1321 aagggtacta gcgctgatcg gcaattgcga ggtcctagga cgatctctat cactgatagg
     1381 gagatctcta tcactgatag ggagagctct gcttatatag acctcccacc gtacacgcct
     1441 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
     1501 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     1561 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
     1621 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     1681 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     1741 atacacttga tgtactctag aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     1801 ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat
     1861 ctccctatca gtgatagaga tcgtcaagct tggtaccgag ctcggatccg aaaacctgta
     1921 cttccaagga accggtggtc acctgtacaa tcgatcctgc aggagctagc gagaatttgt
     1981 attttcaggg tgatatcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2041 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc ggcgagggcg
     2101 agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2161 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cgtggcctgc ttcagccgct
     2221 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2281 aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc gaggtgaagt
     2341 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2401 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccactacgtc tatatcacgg
     2461 ccgacaagca gaagaactgc atcaaggcta acttcaagat ccgccacaac gttgaggacg
     2521 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2581 tgctgcccga caaccactac ctgagccatc agtccaagct gagcaaagac cccaacgaga
     2641 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggattaca catggcatgg
     2701 acgagctgta caagtaactc gaggtttaaa cccgctgatc agcctcgact gtgccttcta
     2761 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2821 ctcccactgt cctttcctaa taaaatgagg aaattgtatc gcattgtctg agtaggtgtc
     2881 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2941 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3001 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3061 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3121 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3181 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3241 atggttcacg tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa
     3301 cttccttggc caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     3361 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     3421 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     3481 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     3541 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     3601 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     3661 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     3721 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     3781 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     3841 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     3901 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     3961 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     4021 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     4081 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     4141 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     4201 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     4261 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     4321 gggcaaagga atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     4381 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4441 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4501 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4561 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4621 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4681 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4741 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4801 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4861 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4921 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4981 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5041 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5101 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5161 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5221 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5281 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5341 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5401 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5461 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5521 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5581 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5641 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5701 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5761 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6781 cacctgacgt cgacggatc
//
