LOCUS       sequence-183029-        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 G3.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    101..122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(137..142,143..160,161..167)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    175..191
                     /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     180..202
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     199..215
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     199..215
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     234..254
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        236..254
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        282..508
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             509..689
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    736..861
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1354..1587
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1772..1816
                     /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        1994..2242
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1994..2014
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2165..2184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2397..2514
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2583..2794
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2820..3419
                     /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(2820..2839)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3316..3336
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3435..4023
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3488..3508)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3906..3917)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4095..4328
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4406..4527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4443..4462)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4497..4516
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4567..4702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4722..4741)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4723..4741)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4751..4768)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4751..4767)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4760..4782)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4909..5364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4996..5015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5206..5227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5390..5494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5495..5563,5564..6355)
                     /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(5713..5732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6526..7114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7015..7034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7268..7285
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
       61 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      121 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      181 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      241 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      301 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      361 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      421 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      481 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      541 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      601 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      661 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      721 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      781 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      841 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      901 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
      961 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1021 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1081 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1141 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1201 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1261 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1321 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1381 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1441 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1501 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1561 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1621 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1681 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1741 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1801 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1861 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1921 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     1981 gaggggaccc agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2041 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2101 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2161 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2221 cttgtggaaa ggacgaaaca ccgctctgca ggtttacacc gcgtttaaga gctatgctgg
     2281 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     2341 gtcggtgctt tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt
     2401 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2461 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     2521 tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg gtgcccgtca
     2581 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2641 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2701 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     2761 tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga tccgccacca
     2821 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     2881 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     2941 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3001 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     3061 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     3121 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     3181 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     3241 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     3301 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     3361 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     3421 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3481 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3541 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3601 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3661 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3721 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3781 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     3841 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3901 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     3961 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     4021 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     4081 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     4141 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4201 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4261 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4321 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     4381 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     4441 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4501 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     4561 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4621 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4681 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     4741 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4801 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4861 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     4921 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4981 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5041 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5101 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     5161 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5221 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     5281 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5341 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5401 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5461 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5521 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5581 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5641 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5701 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5761 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5821 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5881 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5941 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6001 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6061 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6121 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6181 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6241 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6301 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6361 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6421 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6481 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6541 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6601 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6661 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6721 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6781 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6841 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6901 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6961 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7021 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7081 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7141 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7201 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     7261 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc c
//
