LOCUS       sequence-234914-        6693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses dox-inducible PPP2R5E resistant to siRNA pool 2 used in 
            manuscript.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6693)
  AUTHORS   Lee SJ, Rodriguez-Bravo V, Kim H, Datta S, Foley EA
  TITLE     The PP2A(B56) phosphatase promotes the association of Cdc20 with 
            APC/C in mitosis.
  JOURNAL   J Cell Sci. 2017 May 15;130(10):1760-1771. doi: 10.1242/jcs.201608. 
            Epub 2017 Apr 12.
  PUBMED    28404789
REFERENCE   2  (bases 1 to 6693)
  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"; doi: 
            "10.1242/jcs.201608"; journalName: "J Cell Sci"; date: "2017-05-15- 
            15"; volume: "130"; issue: "10"; pages: "1760-1771"
COMMENT     Sequence Label: FLAG-PPP2R5E siRNA-resistant
FEATURES             Location/Qualifiers
     source          1..6693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             984..1007
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1023..1043
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1056..1100
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    1134..1158
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2569..2593)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2645..2662)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2651..2875
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3008..3027)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3159..3206
                     /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             3214..4236
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3214..4236
                     /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    4366..4487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4403..4422)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4457..4476
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4536..4552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4536..4552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4549..4571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4560..4576
                     /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(4584..4590,4591..4608,4609..4614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4629..4650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4767..4784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4938..5526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5018..5037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5697..6488,6489..6557))
                     /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     6320..6339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6558..6662)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag atggattaca aggatgacga cgataaggca ggcgggggcg
     1021 gggagaatct ttattttcag ggcggcgggg gcgggaaaga aaccgctgct gctaaattcg
     1081 aacgccagca catggacagc ggaggtggag gtattaacta taacggtcat caaacaagtt
     1141 tgtacaaaaa agcaggctcc accatggggt cctcagcacc aactactcct ccatcagtgg
     1201 ataaagtaga cggattttct cggaagtccg tcagaaaagc cagacagaag aggtcgcaaa
     1261 gttcctcaca gtttaggtct caaggcaagc ctattgagtt aacacctctg ccgctgctaa
     1321 aagacgttcc atcctcagag cagcctgaac tgttcctaaa gaaacttcag cagtgctgtg
     1381 tcatttttga cttcatggac acgctatctg atcttaaaat gaaagaatac aagcgctcca
     1441 ctcttaatga actggtggac tacattacaa taagcagagg ctgtttgaca gagcagactt
     1501 accctgaagt agttagaatg gtatcttgca atatattcag aactctccct cctagtgaca
     1561 gcaatgaatt tgatccagaa gaagatgaac ctacccttga ggcatcgtgg ccacacttac
     1621 agcttgtata tgaatttttc atacgatttt tggaaagcca agaattccaa cccagcattg
     1681 ccaaaaaata tatagatcag aaatttgtat tacagcttct ggagctattt gacagcgaag
     1741 accctcggga acgggactac ttaaaaacag tcttacacag aatttatggc aagtttcttg
     1801 gtcttagagc atttatccga aaacagatta acaatatttt tctaaggttt gtttatgaaa
     1861 cagaacactt caatggtgta gctgaactgc tggaaatatt aggaagtatt atcaatggct
     1921 ttgctttacc tcttaaggca gaacacaaac agtttctggt gaaagtattg atccctttac
     1981 acactgtcag gagcttatca ctcttccatg cccaattagc atattgtata gtacagtttc
     2041 tggagaaaga tccttcactc acagaaccag ttattagggg gttaattaaa ttttggccta
     2101 aaacatgtag tcaaaaagag gtcatgttcc ttggggaact ggaagaaata ttggatgtga
     2161 ttgaaccttc acaatttgtt aaaatccaag aacctttgtt taaacaaatc gccaagtgtg
     2221 tatctagccc ccattttcag gtggcagaaa gagcactcta ttattggaat aatgaataca
     2281 tcatgagttt gatagaagaa aactctaacg tcatccttcc catcatgttt tccagccttt
     2341 ataggatttc aaaagagcat tggaatccgg ctattgtggc gttggtgtac aatgtgttga
     2401 aggcatttat ggaaatgaac agcaccatgt ttgacgagct gacagccaca tacaagtcag
     2461 atcgtcagcg tgagaaaaag aaagaaaagg agcgtgaaga attgtggaaa aaattggagg
     2521 atctggagtt aaagagaggt cttagacgtg atggaataat tccaactgac ccagctttct
     2581 tgtacaaagt ggttcgatca gggtaatctc gagtctagag ggcccgttta aacccgctga
     2641 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2701 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2761 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2821 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2881 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2941 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3001 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3061 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3121 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     3181 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     3241 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     3301 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     3361 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     3421 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     3481 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     3541 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     3601 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     3661 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     3721 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3781 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3841 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3901 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3961 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     4021 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     4081 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     4141 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     4201 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     4261 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4321 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4381 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4441 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4501 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4561 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4621 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4681 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4741 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4801 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4861 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4921 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4981 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5041 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5101 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5161 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5221 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5281 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5341 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5401 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5461 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5521 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5581 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5641 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5701 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5761 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5821 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5881 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5941 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6001 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6061 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6121 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6181 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6241 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6301 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6361 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6421 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6481 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6541 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6601 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6661 cgcacatttc cccgaaaagt gccacctgac gtc
//
