LOCUS       sequence-203365-        5903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of your protein of interest in fusion with green 
            fluorescent protein at the N-terminus (cleavable by TEV), (PMID: 
            8885998, 8805248). EGFP is not truly monomeric (PMID: 11988576, 
            22869113).
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 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-EGFP-TEV
FEATURES             Location/Qualifiers
     source          1..5903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..53)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        225..604
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        605..808
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     759..779
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    810..828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    831..849
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     859..883
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(980..982,983..985,986..1696)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(980..982,983..985,986..1696)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1025..1046)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1286..1305)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1633..1654
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1703..1723
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1764..1784
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1845..1862)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1851..2075
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2208..2227)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2359..2406
                     /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             2414..3436
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2414..3436
                     /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    3566..3687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3603..3622)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3657..3676
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3736..3752)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3736..3752)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3749..3771)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3760..3776
                     /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(3784..3790,3791..3808,3809..3814))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3829..3850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3967..3984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4138..4726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4218..4237)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4897..5688,5689..5757))
                     /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     5520..5539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5758..5862)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
       61 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
      121 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      181 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      781 acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc tccctatcag
      841 tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc
      901 acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga ctctggcgtt
      961 taaacttaag cttggtacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1021 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1081 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1141 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1201 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1261 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1321 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1381 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     1441 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1501 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     1561 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     1621 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     1681 ggacgagctg tacaagggat ccgaaaacct gtacttccaa ggaaccggtg gtcacctgta
     1741 caatcgatcc tgcaggagct agcgagaatt tgtattttca gggtgatatc cagcacagtg
     1801 gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     1861 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     1921 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     1981 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2041 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2101 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2161 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2221 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2281 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2341 tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg
     2401 aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     2461 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     2521 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     2581 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     2641 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     2701 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     2761 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     2821 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     2881 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     2941 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     3001 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     3061 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     3121 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     3181 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     3241 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     3301 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     3361 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     3421 gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag
     3481 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     3541 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3601 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3661 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     3721 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3781 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3841 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3901 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3961 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4021 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4081 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4141 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4201 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4261 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4321 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4381 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4441 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4501 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4561 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4621 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4681 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4741 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4801 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4861 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4921 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4981 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5041 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5101 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5161 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5221 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5281 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5341 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5401 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5461 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5521 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5581 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5641 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5701 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5761 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5821 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5881 gccacctgac gtcgacggat cgg
//
