LOCUS       sequence-203370-        5918 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: 
            24633408). mCardinal is suitable for deep-tissue imaging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  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-mCardinal-TEV
FEATURES             Location/Qualifiers
     source          1..5918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..56)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        228..607
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        608..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     762..782
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    813..831
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    834..852
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     862..886
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             983..1714
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             1721..1741
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1782..1802
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1863..1880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1869..2093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2226..2245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2377..2424
                     /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             2432..3454
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2432..3454
                     /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    3584..3705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3621..3640)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3675..3694
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3754..3770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3754..3770)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3767..3789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3778..3794
                     /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(3802..3808,3809..3826,3827..3832))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3847..3868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3985..4002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4156..4744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4236..4255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4915..5706,5707..5775))
                     /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     5538..5557
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5776..5880)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
       61 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
      121 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
      181 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
      241 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
      301 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      361 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      421 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      481 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      541 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      601 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      661 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      721 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      781 tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag atctccctat
      841 cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
      901 tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc ggactctggc
      961 gtttaaactt aagcttggta ccatggtgag caagggcgag gagctgatca aggagaacat
     1021 gcacatgaag ctgtacatgg aaggcaccgt gaacaaccac cacttcaagt gcaccaccga
     1081 aggggagggc aagccctacg agggcaccca gacccagagg attaaggtgg tggagggagg
     1141 ccccctgccg ttcgcattcg acatcctggc cacctgcttt atgtacggga gcaagacctt
     1201 catcaaccac acccagggca tccccgattt ctttaagcag tccttccctg agggcttcac
     1261 atgggagaga gtcaccacat acgaagacgg gggcgtgctt accgttaccc aggacaccag
     1321 cctccaggac ggctgcttga tctacaacgt caagctcaga ggggtgaact tcccatccaa
     1381 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc accaccgaga ccctgtaccc
     1441 cgctgacggc ggcctggaag gcagatgcga catggccctg aagctcgtgg gcgggggcca
     1501 cctgcactgc aacctgaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
     1561 gcccggcgtc tactttgtgg accgcagact ggaaagaatc aaggaggccg acaatgagac
     1621 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
     1681 gcacaaactt aatggcatgg acgagctgta caagggatcc gaaaacctgt acttccaagg
     1741 aaccggtggt cacctgtaca atcgatcctg caggagctag cgagaatttg tattttcagg
     1801 gtgatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     1861 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1921 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1981 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2041 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2101 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2161 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2221 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2281 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2341 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     2401 ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg cgacgtctgt
     2461 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     2521 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     2581 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     2641 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     2701 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     2761 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     2821 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     2881 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     2941 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     3001 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     3061 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     3121 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     3181 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     3241 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     3301 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     3361 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     3421 acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt tcgattccac
     3481 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     3541 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     3601 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3661 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     3721 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3781 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3841 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3901 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3961 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4021 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4081 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4141 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4201 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4261 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4321 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4381 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4441 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4501 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4561 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4621 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4681 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4741 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4801 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4861 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4921 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4981 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5041 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5101 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5161 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5221 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5281 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5341 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5401 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5461 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5521 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5581 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5641 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5701 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5761 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5821 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5881 cacatttccc cgaaaagtgc cacctgacgt cgacggat
//
