LOCUS       sequence-189885-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TOPBP1.
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: TOPBP1 G2.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    130..151
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(166..171,172..189,190..196)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    204..220
                     /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     209..231
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     228..244
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     228..244
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     263..283
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        265..283
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        311..537
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             538..718
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    765..890
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1383..1616
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1801..1845
                     /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        2023..2271
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2023..2043
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2194..2213
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2427..2544
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2613..2824
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2850..3449
                     /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(2850..2869)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3346..3366
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3465..4053
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3518..3538)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3936..3947)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4125..4358
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4436..4557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4473..4492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4527..4546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4597..4732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4752..4771)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4753..4771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4781..4798)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4781..4797)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4790..4812)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4939..5394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5026..5045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5236..5257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5420..5524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5525..5593,5594..6385)
                     /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(5743..5762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6556..7144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7045..7064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7298..7302,1..13)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
       61 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      121 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
      181 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
      241 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct
      301 gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt
      361 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg
      421 tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc
      481 actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg
      541 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      601 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      661 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      721 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      781 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      841 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      901 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
      961 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1021 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1081 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1141 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1201 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1261 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1321 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1381 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1441 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1501 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1561 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1621 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1681 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1741 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1801 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1861 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1921 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     1981 ttatcgtttc agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg
     2041 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     2101 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     2161 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     2221 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtatatctg
     2281 tgaccctttt aggtttaaga gctatgctgg aaacagcata gcaagtttaa ataaggctag
     2341 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt ctagatcttg
     2401 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     2461 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     2521 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     2581 cgccggctcg agtggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2641 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
     2701 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2761 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2821 acaggtgtcg tgacgcggga tccgccacca tgaccgagta caagcccacg gtgcgcctcg
     2881 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     2941 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     3001 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     3061 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     3121 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     3181 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     3241 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     3301 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     3361 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     3421 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt
     3481 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3541 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3601 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3661 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3721 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3781 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3841 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     3901 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3961 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4021 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     4081 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     4141 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     4201 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     4261 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     4321 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     4381 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     4441 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4501 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4561 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4621 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4681 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     4741 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     4801 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4861 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4921 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4981 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5281 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5341 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5521 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6061 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6241 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6361 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6541 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6601 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6661 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6721 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6781 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6841 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6901 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6961 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7021 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7081 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7141 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7201 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7261 agctgatacc gctcgccgca gccgaacgac cgagcgcagc ga
//
