LOCUS       Exported                6597 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Functional studies using RNAi; rescue experiments; substituting 
            protein for its different form. miRNA cassette with nuclear mCherry 
            expression marker; your protein of interest with C-terminal FLAG..
ACCESSION   .
VERSION     .
KEYWORDS    pKK-RNAi-nucCHERRYmiR-TEV-FLAG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6597)
  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 6597)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6597
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(16..36)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    431..479
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     504..523
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     ncRNA           complement(595..636)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(700..726)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(733..774)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(838..864)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(871..912)
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(976..1002)
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     CDS             complement(1048..1068)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1072..1092)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1096..1116)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1129..1836)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1404..1423)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1673..1691
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     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"
     promoter        complement(1891..2094)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        2274..2325
                     /label=CMVd2 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields moderately reduced expression 
                     relative to the full-length promoter"
     protein_bind    2327..2345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2348..2366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             2402..2422
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2463..2483
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2490..2513
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2542..2559)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2548..2772
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2905..2924)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3056..3103
                     /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             3111..4133
                     /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    4263..4384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4300..4319)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4354..4373
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4433..4449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4433..4449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4446..4468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4457..4473
                     /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(4481..4511)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4526..4547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4664..4681)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4835..5423)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4915..4934)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5594..6454)
                     /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     6217..6236
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6455..6559)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgggagatcc gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
       61 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
      121 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
      181 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
      241 tccatcgttc agatccttat cgatgctatg gcagggcctg ccgccccgac gttggctgcg
      301 agccctgggc cttcacccga acttgggggg tggggtgggg aaaaggaaga aacgcgggcg
      361 tattggcccc aatggggtca cggtggggta tcgacagagt gccagccctg ggaccgaacc
      421 ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt
      481 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatca
      541 gcgaaccgcg ggccctctag atcactagat atgacgcgtg cggccgcaga tctgggccat
      601 ttgttccatg tgagtgctag taacaggcct tgtgtcctga aatgtactgc gtggagacgt
      661 cagtcagtgg ccaaaacgtc tccacgcgca gtacatttca gcatacagcc ttcagcaagc
      721 ctccaggatc tgggccattt gttccatgtg agtgctagta acaggccttg tgtcctgaaa
      781 tgtactgcgt ggagacgtca gtcagtggcc aaaacgtctc cacgcgcagt acatttcagc
      841 atacagcctt cagcaagcct ccaggatctg ggccatttgt tccatgtgag tgctagtaac
      901 aggccttgtg tcctgaaatg tactgcgtgg agacgtcagt cagtggccaa aacgtctcca
      961 cgcgcagtac atttcagcat acagccttca gcaagcctcc aggatcactt aagactagcg
     1021 ctccaattgc actacctcct cttactacac cttgcgtttc ttctttgggt cgacctttct
     1081 cttcttttta gggtccacct tccgcttttt cttggggtct gctgcggcct tgtacagctc
     1141 gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta
     1201 gtcctcgttg tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg
     1261 cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt
     1321 cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc
     1381 ggaggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt
     1441 ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt
     1501 cacggtcacc acgccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg
     1561 gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc
     1621 gtacatgaac tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac
     1681 cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc
     1741 gaactcgtgg ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat
     1801 gatggccatg ttatcctcct cgcccttgct caccatggtc ctaggacgat ctctatcact
     1861 gatagggaga tctctatcac tgatagggag agctctgctt atatagacct cccaccgtac
     1921 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     1981 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     2041 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     2101 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     2161 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     2221 catatgatac acttgatgta ctctagaaaa tgtcgtaaca actccgcccc attgacgcaa
     2281 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat
     2341 agagatctcc ctatcagtga tagagatcgt cggatctaag cttggtaccg agctcggatc
     2401 cgaaaacctg tacttccaag gaaccggtgg tcacctgtac aatcgatcct gcaggagcta
     2461 gcgagaattt gtattttcag ggtgatatcg attacaagga tgacgacgat aagtagctcg
     2521 aggtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     2581 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     2641 aaaatgagga aattgtatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     2701 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     2761 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     2821 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2881 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2941 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3001 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt
     3061 tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg
     3121 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3181 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3241 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3301 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     3361 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3421 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     3481 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3541 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     3601 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     3661 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     3721 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     3781 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     3841 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     3901 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     3961 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4021 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4081 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac
     4141 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4201 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4261 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4321 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4381 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4441 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4501 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4561 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4621 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4681 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4741 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4801 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4861 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4921 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4981 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5041 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5101 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5161 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5221 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5281 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5341 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5401 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5461 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5521 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5581 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5641 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5701 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5761 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5821 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5881 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5941 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6001 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6061 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6121 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6181 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6241 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6301 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6361 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6421 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6481 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6541 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggat
//
