LOCUS       Exported                6800 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Concomitant expression of two proteins. One protein expressed with 
            mRuby3, cleavable by 3C; second protein expressed with mClover3, 
            cleavable by TEV protease; tags positions: C-termini..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-BI16-ORF1-3C-mRuby3_ORF2-TEV-mClover3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6800)
  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 6800)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6800
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(23..43)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    438..486
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     511..530
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(565..1275)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             complement(1276..1299)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    1372..1390
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1393..1411
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(1413..1616)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        1797..1848
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    1850..1868
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1871..1889
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1919..1939
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1980..2000
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2007..2726
                     /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(2052..2073)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2660..2681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2752..2769)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2758..2982
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3115..3134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3266..3313
                     /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             3321..4343
                     /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    4473..4594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4510..4529)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4564..4583
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4643..4659)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4643..4659)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4656..4678)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4667..4683
                     /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(4691..4721)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4736..4757
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4874..4891)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5045..5633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5125..5144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5804..6664)
                     /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     6427..6446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6665..6769)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatccgct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
       61 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
      121 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      181 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
      241 tctccgctcc atcgttcaga tccttatcga tgctatggca gggcctgccg ccccgacgtt
      301 ggctgcgagc cctgggcctt cacccgaact tggggggtgg ggtggggaaa aggaagaaac
      361 gcgggcgtat tggccccaat ggggtcacgg tggggtatcg acagagtgcc agccctggga
      421 ccgaaccccg cgtttatgaa caaacgaccc aacacccgtg cgttttattc tgtcttttta
      481 ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca gttagcctcc
      541 cccatcagcg aaccgcgggc cttacttgta cagctcgtcc atgccaccac caaggttgct
      601 gtatttggca actgccactt ctctttgcac tacgtaggtt tcattgtcac tctcctcgat
      661 cctttccagg cggtgatcaa cggcatggac accgggcatc ttgatgttcc cgacggtctt
      721 ttttgacctg taagttgtca cgaagttgca gtgcagatgg ccaccaccat caactttcag
      781 tgcgatgtca gtgtatcctc tcagaccacc atctgctgga tacatcatct ctgtattagg
      841 ctcccaaccc ttggtcttct tctgcatcac gggaccattg gagggaaagt ttacccctct
      901 gaccttgacg ttgtagacga gctcgccatc ctcaaggctg gtgtcctgcg tgacggtgac
      961 gactccacca tcttcgtatc tcgtaactct ttcccaagta aaaccctcag gaaaggactg
     1021 tttaaagaaa tcagggatgt cggccgggta cttgataaag gtacggctgc catacatgaa
     1081 cgacgtggca agaatgtcaa aggcaaatgg caggggtcct ccctcgatga ctttgatcct
     1141 catggtttgc actccctcgt acggtctgcc ttctccttca cctgtgcatt tgaattggtg
     1201 gccgttgacc gaaccttcca tgaccacctt catacgcata ttttccttga tcagctcttc
     1261 gcccttagac accatgggcc cctggaacag aacttccaga cgcgtttcag agcggccgca
     1321 ctgatcttaa gggtactagc gctgatcggc aattgcgagg tcctaggacg atctctatca
     1381 ctgataggga gatctctatc actgataggg agagctctgc ttatatagac ctcccaccgt
     1441 acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc
     1501 cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat
     1561 ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg
     1621 gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg
     1681 tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt
     1741 ggcatatgat acacttgatg tactctagaa aaatgtcgta acaactccgc cccattgacg
     1801 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
     1861 gatagagatc tccctatcag tgatagagat cgtcaagctt ggtaccgagc tcggatccga
     1921 aaacctgtac ttccaaggaa ccggtggtca cctgtacaat cgatcctgca ggagctagcg
     1981 agaatttgta ttttcagggt gatatcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2041 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg
     2101 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     2161 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtggcctgct
     2221 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2281 gctacgtcca ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg
     2341 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2401 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cactacgtct
     2461 atatcacggc cgacaagcag aagaactgca tcaaggctaa cttcaagatc cgccacaacg
     2521 ttgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2581 gccccgtgct gctgcccgac aaccactacc tgagccatca gtccaagctg agcaaagacc
     2641 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggattacac
     2701 atggcatgga cgagctgtac aagtaactcg aggtttaaac ccgctgatca gcctcgactg
     2761 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2821 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2881 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2941 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3001 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3061 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3121 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3181 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3241 attagggtga tggttcacgt acctagaagt tcctattccg aagttcctat tctctagaaa
     3301 gtataggaac ttccttggcc aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     3361 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     3421 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     3481 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3541 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     3601 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3661 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3721 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     3781 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3841 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3901 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3961 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     4021 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     4081 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     4141 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     4201 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     4261 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     4321 ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt cgattccacc gccgccttct
     4381 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4441 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4501 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4561 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4621 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4681 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4741 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4801 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4861 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4921 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4981 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5041 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5101 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5161 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5221 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5281 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5341 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5401 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5461 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5521 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5581 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5641 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5701 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5761 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5821 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5881 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5941 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6001 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6061 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6121 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6181 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6241 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6301 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6361 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6421 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6481 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6541 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6601 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6661 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6721 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6781 gaaaagtgcc acctgacgtc
//
