LOCUS       sequence-200358-        6060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Concomitant expression of two proteins. One protein is expressed 
            with mCherry fusion tag (at the C-terminus), cleavable by TEV 
            protease; second tag introduced by user..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6060)
  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 6060)
  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-TEV-mCherry
FEATURES             Location/Qualifiers
     source          1..6060
                     /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"
     protein_bind    641..659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    662..680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(682..885)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1066..1117
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1119..1137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1140..1158
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1188..1208
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1249..1269
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1276..1986
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1421..1439)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1689..1708
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2012..2029)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2018..2242
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2375..2394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2526..2573
                     /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             2581..3603
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2581..3603
                     /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    3733..3854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3770..3789)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3824..3843
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3903..3919)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3903..3919)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3916..3938)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3927..3943
                     /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(3951..3957,3958..3975,3976..3981))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3996..4017
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4134..4151)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4305..4893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4385..4404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5064..5855,5856..5924))
                     /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     5687..5706
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5925..6029)
                     /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 ccttatctac gcgtttcaga gcggccgcac tgatcttaag
      601 ggtactagcg ctgatcggca attgcgaggt cctaggacga tctctatcac tgatagggag
      661 atctctatca ctgataggga gagctctgct tatatagacc tcccaccgta cacgcctacc
      721 gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg
      781 gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc
      841 aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg taatagcgat
      901 gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt actgggcata
      961 atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata
     1021 cacttgatgt actctagaaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1081 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
     1141 ccctatcagt gatagagatc gtcaagcttg gtaccgagct cggatccgaa aacctgtact
     1201 tccaaggaac cggtggtcac ctgtacaatc gatcctgcag gagctagcga gaatttgtat
     1261 tttcagggtg atatcatggt gagcaagggc gaggaggata acatggccat catcaaggag
     1321 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     1381 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1441 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     1501 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     1561 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     1621 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     1681 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     1741 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     1801 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     1861 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     1921 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     1981 aagtagctcg aggtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2041 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2101 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2161 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2221 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2281 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2341 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2401 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2461 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2521 acctagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc
     2581 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     2641 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     2701 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     2761 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     2821 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     2881 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     2941 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     3001 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     3061 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     3121 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     3181 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     3241 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     3301 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     3361 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     3421 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     3481 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     3541 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     3601 tagcacgtac tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3661 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3721 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3781 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3841 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3901 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3961 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4021 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4081 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4141 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4201 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4261 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4321 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4381 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4441 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4501 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4561 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4621 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4681 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4741 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4801 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4861 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4921 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4981 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5041 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5101 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5161 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5221 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5281 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5341 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5401 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5461 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5521 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5581 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5641 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5701 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5761 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5821 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5881 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5941 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6001 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
