LOCUS       sequence-336095-        7241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of 3x-Flag-tagged human PP1 alpha catalytic 
            subunit H248K mutant that loses enzymatic activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  AUTHORS   Liu X, Han W, Gulla S, Simon NI, Gao Y, Cai C, Yang H, Zhang X, Liu 
            J, Balk SP, Chen S
  TITLE     Protein phosphatase 1 suppresses androgen receptor ubiquitylation 
            and degradation.
  JOURNAL   Oncotarget. 2016 Jan 12;7(2):1754-64. doi: 10.18632/oncotarget.6434.
  PUBMED    26636645
REFERENCE   2  (bases 1 to 7241)
  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: 
            "10.18632/oncotarget"; journalName: "Oncotarget"; date: "2016-01-12-
            12"; volume: "7"; issue: "2"; pages: "1754-64"
COMMENT     Sequence Label: Flag-neo-PP1-Alpha-H248K
FEATURES             Location/Qualifiers
     source          1..7241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             931..996
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2000..2622
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2199..2218)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2646..2666)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2664..2961
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3015..3809
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3069..3088)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3679..3698
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4461..4595
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4498..4517)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4552..4571
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4600..4619)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4601..4619)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4633..4649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4633..4649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4646..4668)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4657..4673
                     /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(4681..4687,4688..4705,4706..4711))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4726..4747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4864..4881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5035..5623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5115..5134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5794..6585,6586..6654))
                     /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     6417..6436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6655..6759)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6786..7214
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6873..6892)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7083..7104
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagctta tgtccgacag cgagaagctc
     1021 aacctggact cgatcatcgg gcgcctgctg gaagtgcagg gctcgcggcc tggcaagaat
     1081 gtacagctga cagagaacga gatccgcggt ctgtgcctga aatcccggga gatttttctg
     1141 agccagccca ttcttctgga gctggaggca cccctcaaga tctgcggtga catacacggc
     1201 cagtactacg accttctgcg actatttgag tatggcggtt tccctcccga gagcaactac
     1261 ctctttctgg gggactatgt ggacaggggc aagcagtcct tggagaccat ctgcctgctg
     1321 ctggcctata agatcaagta ccccgagaac ttcttcctgc tccgtgggaa ccacgagtgt
     1381 gccagcatca accgcatcta tggtttctac gatgagtgca agagacgcta caacatcaaa
     1441 ctgtggaaaa ccttcactga ctgcttcaac tgcctgccca tcgcggccat agtggacgaa
     1501 aagatcttct gctgccacgg aggcctgtcc ccggacctgc agtctatgga gcagattcgg
     1561 cggatcatgc ggcccacaga tgtgcctgac cagggcctgc tgtgtgacct gctgtggtct
     1621 gaccctgaca aggacgtgca gggctggggc gagaacgacc gtggcgtctc ttttaccttt
     1681 ggagccgagg tggtggccaa gttcctccac aagcacgact tggacctcat ctgccgagca
     1741 aagcaggtgg tagaagacgg ctacgagttc tttgccaagc ggcagctggt gacacttttc
     1801 tcagctccca actactgtgg cgagtttgac aatgctggcg ccatgatgag tgtggacgag
     1861 accctcatgt gctctttcca gatcctcaag cccgccgaca agaacaaggg gaagtacggg
     1921 cagttcagtg gcctgaaccc tggaggccga cccatcaccc caccccgcaa ttccgccaaa
     1981 gccaagaaat agggatcccg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc
     2041 cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
     2101 tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa
     2161 ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag
     2221 tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct
     2281 gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt
     2341 gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
     2401 tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc
     2461 tcccttccct gtccttctga ttttaaaata actataccag caggaggacg tccagacaca
     2521 gcataggcta cctggccatg cccaaccggt gggacatttg agttgcttgc ttggcactgt
     2581 cctctcatgc gttgggtcca ctcagtagat gcctgttgaa ttgggtacgc ggccagcttg
     2641 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     2701 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     2761 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     2821 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2881 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2941 gtagtgagga ggcttttttg gaggaattga tcagcttggg atctgatcaa gagacaggat
     3001 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     3061 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3121 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3181 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3241 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3301 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3361 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3421 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3481 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3541 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3601 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3661 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3721 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3781 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     3841 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3901 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3961 gctggagttc ttcgcccacc ccgggctcga tcccctcgcg agttggttca gctgctgcct
     4021 gaggctggac gacctcgcgg agttctaccg gcagtgcaaa tccgtcggca tccaggaaac
     4081 cagcagcggc tatccgcgca tccatgcccc cgaactgcag gagtggggag gcacgatggc
     4141 cgctttggtc gacccggacg ggacgctcct gcgcctgata cagaacgaat tgcttgcagg
     4201 catctcatga gtgtgtcttc ccgttttccg cctgaggtca ctgcgtggat ggagcgctgg
     4261 cgcctgctgc gcgacggcga gctgctcacc acccactcgc caagctggaa ccgtaaaaag
     4321 gccgcgttgc tggcgttttt ccataggctc cgccgatcat aatcagccat accacatttg
     4381 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     4441 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     4501 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4561 ccaaactcat caatgtatct tatcatgtct ggatcaattc cctatagtga gtcgtattaa
     4621 attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4681 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4741 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4801 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     4861 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4921 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4981 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5041 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5101 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5161 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5221 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5281 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5341 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5401 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5461 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5521 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5581 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5641 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5701 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5761 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5821 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5881 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5941 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6001 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6061 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6121 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6181 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6241 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6301 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6361 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6421 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6481 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6541 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6601 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6661 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6721 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6781 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6841 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6901 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6961 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     7021 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7081 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7141 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7201 atttaacgcg aattttaaca aaatattaac gcttacaatt t
//
