LOCUS       Exported                5877 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of your protein of interest in fusion with blue 
            fluorescent protein at the C-terminus (cleavable by TEV) (PMID: 
            18940671, 25378625). Useful for multi-channel live cell imaging..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-TEV-mTagBFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5877)
  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 5877)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5877
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    824..842
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    845..863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     873..897
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1006..1026
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1067..1087
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1094..1795
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1833..1850)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1839..2063
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2196..2215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2347..2394
                     /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             2402..3424
                     /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    3554..3675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3591..3610)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3645..3664
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3724..3740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3724..3740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3737..3759)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3748..3764
                     /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(3772..3802)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3817..3838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3955..3972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4126..4714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4206..4225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4885..5745)
                     /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     5508..5527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5746..5850)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga
      841 gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc
      901 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
      961 cggactctgg cgtttaaact taagcttggt accgagctcg gatccgaaaa cctgtacttc
     1021 caaggaaccg gtggtcacct gtacaatcga tcctgcagga gctagcgaga atttgtattt
     1081 tcagggtgat atcatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     1141 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     1201 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     1261 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     1321 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     1381 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     1441 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     1501 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     1561 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     1621 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     1681 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     1741 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attaactcga
     1801 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     1861 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     1921 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     1981 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2041 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2101 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2161 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2221 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2281 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2341 tacctagaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc
     2401 caaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag
     2461 cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt
     2521 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg
     2581 ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
     2641 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca
     2701 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc
     2761 gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat
     2821 cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
     2881 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
     2941 gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
     3001 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat
     3061 gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg
     3121 tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
     3181 gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
     3241 caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc
     3301 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
     3361 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga
     3421 atagcacgta ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     3481 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     3541 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3601 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3661 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     3721 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     3781 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     3841 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     3901 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     3961 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4021 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4081 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4141 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4201 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4261 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4321 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4381 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4441 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4501 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4561 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4621 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4681 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4741 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     4801 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4861 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4921 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4981 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5041 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5101 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5161 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5221 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     5281 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5341 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5401 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5461 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     5521 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5581 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5641 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5701 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5761 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5821 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctga
//
