LOCUS       Exported                5910 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of your protein of interest in fusion with red 
            fluorescent protein at the C-terminus (cleavable by TEV), (PMID: 
            24633408). mCardinal is suitable for deep-tissue imaging..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-TEV-mCardinal
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5910)
  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 5910)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5910
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             1002..1022
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1063..1083
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1090..1824
                     /codon_start=1
                     /product="far-red fluorescent protein derived from 
                     mNeptune, with further red-shifted spectra"
                     /label=mCardinal
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTTEGEGKPYEGTQTQ
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEATTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(1862..1879)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1868..2092
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2225..2244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2376..2423
                     /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             2431..3453
                     /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    3583..3704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3620..3639)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3674..3693
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3753..3769)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3753..3769)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3766..3788)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3777..3793
                     /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(3801..3831)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3846..3867
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3984..4001)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4155..4743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4235..4254)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4914..5774)
                     /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     5537..5556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5775..5879)
                     /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 ctctggcgtt taaacttaag cttggtaccg agctcggatc cgaaaacctg tacttccaag
     1021 gaaccggtgg tcacctgtac aatcgatcct gcaggagcta gcgagaattt gtattttcag
     1081 ggtgatatca tggtgagcaa gggcgaggag ctgatcaagg agaacatgca catgaagctg
     1141 tacatggaag gcaccgtgaa caaccaccac ttcaagtgca ccaccgaagg ggagggcaag
     1201 ccctacgagg gcacccagac ccagaggatt aaggtggtgg agggaggccc cctgccgttc
     1261 gcattcgaca tcctggccac ctgctttatg tacgggagca agaccttcat caaccacacc
     1321 cagggcatcc ccgatttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
     1381 accacatacg aagacggggg cgtgcttacc gttacccagg acaccagcct ccaggacggc
     1441 tgcttgatct acaacgtcaa gctcagaggg gtgaacttcc catccaacgg ccctgtgatg
     1501 cagaagaaaa cactcggctg ggaggccacc accgagaccc tgtaccccgc tgacggcggc
     1561 ctggaaggca gatgcgacat ggccctgaag ctcgtgggcg ggggccacct gcactgcaac
     1621 ctgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
     1681 tttgtggacc gcagactgga aagaatcaag gaggccgaca atgagaccta cgtcgagcag
     1741 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caaacttaat
     1801 ggcatggacg agctgtacaa gtaactcgag tctagagggc ccgtttaaac ccgctgatca
     1861 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1921 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1981 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2041 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2101 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     2161 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2221 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2281 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2341 aaaaaacttg attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat
     2401 tctctagaaa gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc
     2461 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     2521 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     2581 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     2641 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     2701 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     2761 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     2821 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     2881 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     2941 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     3001 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     3061 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     3121 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     3181 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     3241 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     3301 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     3361 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     3421 cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc
     3481 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     3541 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     3601 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3661 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3721 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3781 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3841 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3901 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3961 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4021 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4081 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4141 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4201 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4261 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4321 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4381 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4441 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4501 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4561 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4621 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4681 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4741 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4801 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4861 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4921 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4981 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5041 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5101 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5161 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5221 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5281 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5341 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5401 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5461 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5521 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5581 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5641 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5701 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5761 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5821 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5881 acatttcccc gaaaagtgcc acctgacgtc
//
