LOCUS       sequence-203367-        5903 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of your protein of interest in fusion with green 
            fluorescent protein at the N-terminus (cleavable by TEV). mClover3 
            is a brighter derivative of mEGFP (PMID: 26879144)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5903)
  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 5903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mClover3-TEV
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..54)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        226..605
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        606..809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     760..780
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    811..829
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    832..850
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     860..884
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(981..983,984..986,987..1697)
                     /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(1026..1047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1634..1655
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1704..1724
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1765..1785
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1846..1863)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1852..2076
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2209..2228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2360..2407
                     /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             2415..3437
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2415..3437
                     /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    3567..3688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3604..3623)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3658..3677
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3737..3753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3737..3753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3750..3772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3761..3777
                     /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(3785..3791,3792..3809,3810..3815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3830..3851
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3968..3985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4139..4727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4219..4238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4898..5689,5690..5758))
                     /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     5521..5540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5759..5863)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
       61 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
      121 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
      181 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
      241 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      301 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      361 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      421 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      481 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      541 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      601 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      661 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      721 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      781 tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat ctccctatca
      841 gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
      901 cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg actctggcgt
      961 ttaaacttaa gcttggtacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1021 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg
     1081 gcgagggcga tgccaccaac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1141 tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc
     1201 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1261 tccaggagcg caccatctct ttcaaggacg acggtaccta caagacccgc gccgaggtga
     1321 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1381 acggcaacat cctggggcac aagctggagt acaacttcaa cagccactac gtctatatca
     1441 cggccgacaa gcagaagaac tgcatcaagg ctaacttcaa gatccgccac aacgttgagg
     1501 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1561 tgctgctgcc cgacaaccac tacctgagcc atcagtccaa gctgagcaaa gaccccaacg
     1621 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatt acacatggca
     1681 tggacgagct gtacaaggga tccgaaaacc tgtacttcca aggaaccggt ggtcacctgt
     1741 acaatcgatc ctgcaggagc tagcgagaat ttgtattttc agggtgatat ccagcacagt
     1801 ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     1861 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1921 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1981 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2041 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2101 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2161 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2221 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2281 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2341 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     2401 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     2461 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     2521 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     2581 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     2641 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     2701 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     2761 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     2821 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     2881 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2941 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3001 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     3061 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     3121 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     3181 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     3241 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     3301 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     3361 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     3421 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     3481 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3541 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3601 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3661 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3721 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3781 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3841 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3901 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3961 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4021 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4081 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4141 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4201 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4261 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4321 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4381 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4441 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4501 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4561 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4621 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4681 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4741 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4801 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4861 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4921 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4981 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5041 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5101 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5161 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5221 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5281 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5341 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5401 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5461 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5521 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5581 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5641 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5701 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5821 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5881 tgccacctga cgtcgacgga tcg
//
