LOCUS       sequence-183896-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PPP2R5D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7301)
  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: 
            "10.1016/j.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: PPP2R5D B12.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..70
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    187..208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(223..228,229..246,247..253)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    261..277
                     /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)."
     primer_bind     266..288
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     285..301
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     285..301
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     320..340
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        322..340
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        368..594
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             595..775
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    822..947
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1440..1673
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1858..1902
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2080..2328
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2080..2100
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2251..2270
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2483..2600
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2669..2880
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2906..3505
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2906..2925)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3402..3422
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3521..4109
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3574..3594)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3992..4003)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4181..4414
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4492..4613
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4529..4548)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4583..4602
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4653..4788
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4808..4827)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4809..4827)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4837..4854)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4837..4853)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4846..4868)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4995..5450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5082..5101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5292..5313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5476..5580
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5581..5649,5650..6441)
                     /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     complement(5799..5818)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6612..7200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7101..7120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
       61 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      121 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      181 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      241 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      301 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
      361 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
      421 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      481 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      541 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      601 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      661 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      721 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      781 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      841 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      901 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      961 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1021 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1081 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1141 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1201 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1261 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1321 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1381 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1441 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1501 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1561 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1621 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1681 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1741 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1801 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1861 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1921 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1981 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2041 tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt
     2101 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
     2161 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     2221 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     2281 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc tgtgcatttg
     2341 gcaaccttgt ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg
     2401 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctag atcttgagac
     2461 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     2521 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     2581 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc
     2641 ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     2701 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     2761 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2821 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     2881 gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag cccacggtgc gcctcgccac
     2941 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3001 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3061 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3121 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3181 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3241 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     3301 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3361 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3421 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3481 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa
     3541 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3601 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3661 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3721 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3781 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3841 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3901 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     3961 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4021 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4081 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     4141 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4201 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4261 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4321 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4381 agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt
     4441 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     4501 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4561 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4621 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4681 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4741 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     4801 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     4861 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4921 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4981 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5041 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5101 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5161 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5221 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5281 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5341 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5401 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5461 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5521 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5581 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5641 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5701 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5761 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5821 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5881 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5941 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6001 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6061 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6121 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6181 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6241 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6301 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6361 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6421 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6481 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6541 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6601 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6661 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6721 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6781 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6841 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6901 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6961 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7021 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7081 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7141 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7201 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7261 ttctttcctg cgttatcccc tgattctgtg gataaccgta t
//
