LOCUS       sequence-218634-        6134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dual Strep-tagged PP1gamma in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6134)
  AUTHORS   Weith M, Seiler J, van den Boom J, Kracht M, Hulsmann J, Primorac I,
            Del Pino Garcia J, Kaschani F, Kaiser M, Musacchio A, Bollen M, 
            Meyer H
  TITLE     Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives
            PP1 Holoenzyme Formation.
  JOURNAL   Mol Cell. 2018 Oct 11. pii: S1097-2765(18)30791-3. doi: 
            10.1016/j.molcel.2018.09.020.
  PUBMED    30344098
REFERENCE   2  (bases 1 to 6134)
  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"; journalName: "Mol Cell. 
            2018 Oct 11. pii: S1097-2765(18)30791-3. doi: 
            10.1016/j.molcel.2018.09.020."
COMMENT     Sequence Label: pcDNA5FRT/TO PP1gamma-2*Strep
FEATURES             Location/Qualifiers
     source          1..6134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    718..736
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    739..757
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     767..791
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1860..1883
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1920..1943
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(1984..2001)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1990..2214
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2347..2366)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2498..2545
                     /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             2553..3575
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2553..3575
                     /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    3705..3826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3742..3761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3796..3815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3875..3891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3875..3891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3888..3910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3899..3915
                     /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(3923..3929,3930..3947,3948..3953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3968..3989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4106..4123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4277..4865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4357..4376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5036..5827,5828..5896))
                     /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     5659..5678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5897..6001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6076..6095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
       61 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      121 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc
      721 ctatcagtga tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga
      781 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg
      841 accgatccag cctccggact ctagcgttta aacttaagat ggcggattta gataaactca
      901 acatcgacag cattatccaa cggctgctgg aagtgagagg gtccaagcct ggtaagaatg
      961 tccagcttca ggagaatgaa atcagaggac tgtgcttaaa gtctcgtgaa atctttctca
     1021 gtcagcctat cctactagaa cttgaagcac cactcaaaat atgtggtgac atccatggac
     1081 aatactatga tttgctgcga ctttttgagt acggtggttt cccaccagaa agcaactacc
     1141 tgtttcttgg ggactatgtg gacaggggaa agcagtcatt ggagacgatc tgcctcttac
     1201 tggcctacaa aataaaatat cctgagaatt tttttcttct cagagggaac catgaatgtg
     1261 ccagcatcaa cagaatttat ggattttatg atgaatgtaa aagaagatac aacattaaac
     1321 tatggaaaac tttcacagac tgttttaact gtttaccgat agcagccatc gtggatgaga
     1381 agatattctg ttgtcatgga ggtttatcac cagatcttca atctatggag cagattcggc
     1441 gaattatgcg accaactgat gtaccagatc aaggtcttct ttgtgatctt ttgtggtctg
     1501 accccgataa agatgtctta ggctggggtg aaaatgacag aggagtgtcc ttcacatttg
     1561 gtgcagaagt ggttgcaaaa tttctccata agcatgattt ggatcttata tgtagagccc
     1621 atcaggtggt tgaagatgga tatgaatttt ttgcaaagag gcagttggtc actctgtttt
     1681 ctgcgcccaa ttattgcgga gagtttgaca atgcaggtgc catgatgagt gtggatgaaa
     1741 cactaatgtg ttcttttcag attttaaagc ctgcagagaa aaagaagcca aatgccacga
     1801 gacctgtaac gcctccaagg ggtatgatca caaagcaagc aaagaaaggc ggaggatctt
     1861 ggagccatcc ccagttcgag aagggcggag gatctggcgg cggaagcggc ggaggaagct
     1921 ggtcccaccc tcagtttgag aaatgactcg agtctagagg gcccgtttaa acccgctgat
     1981 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2041 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2101 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2161 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2221 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2281 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2341 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2401 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2461 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     2521 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     2581 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     2641 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     2701 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     2761 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
     2821 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     2881 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     2941 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     3001 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     3061 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     3121 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     3181 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     3241 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     3301 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     3361 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     3421 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     3481 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     3541 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     3601 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     3721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     3841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     4741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6001 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     6061 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     6121 tgcttgtgtg ttgg
//
