LOCUS       Exported                5895 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of your protein of interest in fusion with green 
            fluorescent protein at the C-terminus (cleavable by TEV). mClover3 
            is a brighter derivative of mEGFP (PMID: 26879144)..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-TEV-mClover3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5895)
  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 5895)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5895
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(49..68)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        240..619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..823
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     774..794
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    825..843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    846..864
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     874..898
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1007..1027
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1068..1088
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1095..1814
                     /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(1140..1161)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1748..1769
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1852..1869)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1858..2082
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2215..2234)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2366..2413
                     /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             2421..3443
                     /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    3573..3694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3610..3629)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3664..3683
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3743..3759)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3743..3759)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3756..3778)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3767..3783
                     /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(3791..3821)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3836..3857
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3974..3991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4145..4733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4225..4244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4904..5764)
                     /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     5527..5546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5765..5869)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
       61 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      121 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      181 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      241 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      301 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      361 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      421 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
      481 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      541 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      601 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      661 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      721 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      781 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag
      841 agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc
      901 ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct
      961 ccggactctg gcgtttaaac ttaagcttgg taccgagctc ggatccgaaa acctgtactt
     1021 ccaaggaacc ggtggtcacc tgtacaatcg atcctgcagg agctagcgag aatttgtatt
     1081 ttcagggtga tatcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1141 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg
     1201 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1261 tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc agccgctacc
     1321 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1381 agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg
     1441 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1501 acatcctggg gcacaagctg gagtacaact tcaacagcca ctacgtctat atcacggccg
     1561 acaagcagaa gaactgcatc aaggctaact tcaagatccg ccacaacgtt gaggacggca
     1621 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1681 tgcccgacaa ccactacctg agccatcagt ccaagctgag caaagacccc aacgagaagc
     1741 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gattacacat ggcatggacg
     1801 agctgtacaa gtaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     1861 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     1921 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     1981 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2041 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     2101 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     2161 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2221 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2281 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2341 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     2401 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     2461 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     2521 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     2581 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     2641 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     2701 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     2761 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     2821 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     2881 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     2941 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3001 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3061 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     3121 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     3181 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     3241 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     3301 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     3361 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     3421 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     3481 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     3541 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     3601 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3661 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     3721 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3781 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3841 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3901 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3961 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4021 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4081 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4141 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4201 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4261 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4321 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4381 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4441 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4501 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4561 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4621 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4681 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4741 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4801 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4861 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4921 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4981 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5041 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5101 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5161 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5221 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5281 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5341 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5401 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5461 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5521 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5581 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5641 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5701 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5761 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5821 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5881 gaaaagtgcc acctg
//
