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). mEGFP is 
            an EGFP A206K mutant with reduced dimerization (PMID: 
            11988576,22869113)..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-TEV-mEGFP
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(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             995..1015
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1056..1076
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1083..1802
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1128..1149)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1389..1408)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1736..1757
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1840..1857)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1846..2070
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2203..2222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2354..2401
                     /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             2409..3431
                     /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    3561..3682
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3598..3617)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3652..3671
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3731..3747)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3731..3747)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3744..3766)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3755..3771
                     /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(3779..3809)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3824..3845
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3962..3979)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4133..4721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4213..4232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4892..5752)
                     /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     5515..5534
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5753..5857)
                     /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 ccgagctcgg atccgaaaac ctgtacttcc aaggaaccgg
     1021 tggtcacctg tacaatcgat cctgcaggag ctagcgagaa tttgtatttt cagggtgata
     1081 tcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1141 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1201 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1261 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1321 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1381 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1441 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1501 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1561 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1621 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1681 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1741 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1801 aactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     1861 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     1921 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     1981 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2041 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2101 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2161 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2221 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2281 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2341 gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     2401 ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     2461 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     2521 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     2581 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     2641 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     2701 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     2761 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     2821 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     2881 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     2941 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     3001 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     3061 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     3121 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     3181 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     3241 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     3301 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     3361 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     3421 caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3481 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3541 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     3601 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3661 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     3721 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3781 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3841 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3901 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3961 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4021 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4081 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4141 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4201 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4261 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4321 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4381 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4441 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4501 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4561 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4621 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4681 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4741 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4801 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4861 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4921 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4981 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5041 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5101 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5161 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5221 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5281 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5341 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5401 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5461 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5521 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5581 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5641 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5701 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5761 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5821 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5881 ctgacgtcga cggat
//
