LOCUS       sequence-190015-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PCNA.
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: PCNA G4.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    81..102
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(117..122,123..140,141..147)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    155..171
                     /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     160..182
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     179..195
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     179..195
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     214..234
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        216..234
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        262..488
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             489..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    716..841
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1334..1567
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1752..1796
                     /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        1974..2222
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1974..1994
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2145..2164
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2377..2494
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2563..2774
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2800..3399
                     /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(2800..2819)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3296..3316
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3415..4003
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3468..3488)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3886..3897)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4075..4308
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4386..4507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4423..4442)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4477..4496
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4547..4682
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4702..4721)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4703..4721)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4731..4748)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4731..4747)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4740..4762)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4889..5344
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4976..4995)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5186..5207
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5370..5474
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5475..5543,5544..6335)
                     /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(5693..5712)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6506..7094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6995..7014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7248..7265
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
       61 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
      121 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
      181 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
      241 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
      301 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
      361 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
      421 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
      481 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
      541 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
      601 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
      661 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
      721 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
      781 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
      841 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
      901 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
      961 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     1021 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1081 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1141 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     1201 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     1261 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     1321 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     1381 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     1441 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     1501 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     1561 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     1621 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     1681 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     1741 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     1801 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     1861 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     1921 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc
     1981 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     2041 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     2101 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     2161 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     2221 ccgttcgagg cgcgcctggt ccgtttaaga gctatgctgg aaacagcata gcaagtttaa
     2281 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt
     2341 ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     2401 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     2461 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     2521 tccactttgg cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc
     2581 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     2641 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2701 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2761 ccgccagaac acaggtgtcg tgacgcggga tccgccacca tgaccgagta caagcccacg
     2821 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     2881 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     2941 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3001 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3061 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3121 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3181 gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3241 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3301 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3361 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa
     3421 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3481 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3541 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3601 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3661 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3721 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3781 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     3841 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3901 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     3961 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     4021 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     4081 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     4141 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     4201 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     4261 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     4321 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     4381 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4441 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4501 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     4561 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4621 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4681 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4741 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4801 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4861 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4921 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4981 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5041 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5101 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5161 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5221 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5281 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5341 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5461 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5521 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5581 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5641 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5701 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5761 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5821 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5881 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5941 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6001 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6061 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6121 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6181 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6241 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6301 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6361 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6421 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6481 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6541 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6601 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6661 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6721 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6781 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6841 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6901 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6961 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7021 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7081 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7141 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7201 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     7261 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct c
//
