LOCUS       sequence-183874-        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 D2.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    54..75
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(90..95,96..113,114..120)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    128..144
                     /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     133..155
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     152..168
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     152..168
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     187..207
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        189..207
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        235..461
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             462..642
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    689..814
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1307..1540
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1725..1769
                     /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        1947..2195
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1947..1967
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2118..2137
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2351..2468
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2537..2748
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2774..3373
                     /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(2774..2793)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3270..3290
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3389..3977
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3442..3462)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3860..3871)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4049..4282
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4360..4481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4397..4416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4451..4470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4521..4656
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4676..4695)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4677..4695)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4705..4722)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4705..4721)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4714..4736)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4863..5318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4950..4969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5160..5181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5344..5448
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5449..5517,5518..6309)
                     /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(5667..5686)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6480..7068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6969..6988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7222..7239
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
       61 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      121 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      181 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      241 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      301 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      361 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      421 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      481 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      541 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      601 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      661 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      721 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      781 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      841 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      901 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      961 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1021 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1081 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1141 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1201 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1261 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1321 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1381 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1441 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1501 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1561 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1621 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1681 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1741 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1801 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1861 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1921 acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc
     1981 atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga
     2041 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2101 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2161 tggctttata tatcttgtgg aaaggacgaa acaccgtggc agtctgtgtc ttgctagttt
     2221 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     2281 aaaagtggca ccgagtcggt gctttttttg aattctagat cttgagacaa atggcagtat
     2341 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2401 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2461 aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc
     2521 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2581 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2641 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2701 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc
     2761 gggatccgcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     2821 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     2881 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     2941 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3001 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3061 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3121 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg
     3181 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3241 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3301 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     3361 gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     3421 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3481 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3541 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3601 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3661 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3721 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3781 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3841 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3901 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3961 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     4021 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     4081 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     4141 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     4201 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     4261 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     4321 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     4381 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4441 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     4501 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4561 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4621 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     4681 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4741 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4801 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4861 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4921 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4981 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5041 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5101 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5161 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5221 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5281 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     5341 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5401 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5461 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5521 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5581 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5641 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5701 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5761 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5821 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5881 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5941 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6001 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6061 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6121 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6181 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6241 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6301 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6361 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6421 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6481 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6541 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6601 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6661 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6721 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6781 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6841 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6901 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6961 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7021 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7081 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7141 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     7201 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     7261 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gc
//
