LOCUS       sequence-203394-        6065 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 6065)
  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 6065)
  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..6065
                     /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        1065..1116
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1118..1136
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1139..1157
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1193..1213
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1254..1274
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1281..1991
                     /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(1426..1444)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1694..1713
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2017..2034)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2023..2247
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2380..2399)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2531..2578
                     /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             2586..3608
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2586..3608
                     /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    3738..3859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3775..3794)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3829..3848
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3908..3924)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3908..3924)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3921..3943)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3932..3948
                     /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(3956..3962,3963..3980,3981..3986))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4001..4022
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4139..4156)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4310..4898)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4390..4409)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5069..5860,5861..5929))
                     /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     5692..5711
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5930..6034)
                     /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 atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1081 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
     1141 cctatcagtg atagagatcg tcggatctaa gcttggtacc gagctcggat ccgaaaacct
     1201 gtacttccaa ggaaccggtg gtcacctgta caatcgatcc tgcaggagct agcgagaatt
     1261 tgtattttca gggtgatatc atggtgagca agggcgagga ggataacatg gccatcatca
     1321 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1381 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     1441 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1501 ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg
     1561 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     1621 aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact
     1681 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc
     1741 ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg
     1801 acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1861 tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca
     1921 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     1981 tgtacaagta gctcgaggtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2041 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2101 ctgtcctttc ctaataaaat gaggaaattg tatcgcattg tctgagtagg tgtcattcta
     2161 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2221 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     2281 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2341 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2401 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2461 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2521 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     2581 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     2641 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     2701 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     2761 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     2821 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     2881 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     2941 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     3001 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     3061 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     3121 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     3181 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     3241 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     3301 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     3361 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     3421 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     3481 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     3541 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     3601 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3661 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3721 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3781 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3841 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     3901 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3961 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4021 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4081 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4141 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4201 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4261 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4321 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4381 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4441 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4501 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4561 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4621 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4681 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4741 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4801 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4861 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4921 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4981 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5041 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5101 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5161 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5221 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5281 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5341 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5401 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5461 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5521 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5581 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5641 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5701 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5761 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5821 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5881 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5941 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6001 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6061 acgtc
//
