LOCUS       sequence-183198-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PBK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  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 7302)
  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: PBK D1.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..68
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    185..206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(221..226,227..244,245..251)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    259..275
                     /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     264..286
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     283..299
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     283..299
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     318..338
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        320..338
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        366..592
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             593..773
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    820..945
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1438..1671
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1856..1900
                     /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        2078..2326
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2078..2098
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2249..2268
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2482..2599
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2668..2879
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2905..3504
                     /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(2905..2924)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3401..3421
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3520..4108
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3573..3593)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3991..4002)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4180..4413
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4491..4612
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4528..4547)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4582..4601
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4652..4787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4807..4826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4808..4826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4836..4853)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4836..4852)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4845..4867)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4994..5449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5081..5100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5291..5312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5475..5579
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5580..5648,5649..6440)
                     /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(5798..5817)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6611..7199
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7100..7119
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
       61 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
      121 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
      181 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
      241 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
      301 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
      361 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
      421 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      481 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      541 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      601 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      661 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      721 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      781 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      841 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      901 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      961 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
     1021 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
     1081 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
     1141 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
     1201 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
     1261 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
     1321 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1381 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1441 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1501 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1561 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1621 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1681 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1741 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1801 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1861 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1921 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1981 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     2041 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     2101 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     2161 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2221 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2281 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgaag acacagactg
     2341 ccatcatgtt taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt
     2401 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattctaga tcttgagaca
     2461 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     2521 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     2581 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg
     2641 gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     2701 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
     2761 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
     2821 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
     2881 tgtcgtgacg cgggatccgc caccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2941 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3001 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3061 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     3121 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3181 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3241 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     3301 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3361 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     3421 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     3481 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     3541 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     3601 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     3661 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     3721 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     3781 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     3841 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     3901 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     3961 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     4021 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     4081 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     4141 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4201 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     4261 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4321 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4381 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     4441 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     4501 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4561 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4621 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4681 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4741 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     4801 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4861 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4921 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4981 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5041 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     5101 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5161 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5221 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5281 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5341 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5401 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5461 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5521 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5581 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5641 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5701 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5761 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5821 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5881 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5941 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6001 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6061 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6121 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6181 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6241 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6301 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6361 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6421 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6481 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6541 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6601 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6661 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6721 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6781 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6841 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6901 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6961 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7021 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7081 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7141 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7201 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7261 tctttcctgc gttatcccct gattctgtgg ataaccgtat ta
//
