LOCUS       sequence-183134-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human TOP2A.
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: TOP2A G12.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     64..81
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    198..219
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(234..239,240..257,258..264)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    272..288
                     /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     277..299
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     296..312
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     296..312
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     331..351
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        333..351
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        379..605
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             606..786
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    833..958
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1451..1684
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1869..1913
                     /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        2091..2339
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2091..2111
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2262..2281
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2494..2611
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2680..2891
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2917..3516
                     /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(2917..2936)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3413..3433
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3532..4120
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3585..3605)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4003..4014)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4192..4425
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4503..4624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4540..4559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4594..4613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4664..4799
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4819..4838)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4820..4838)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4848..4865)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4848..4864)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4857..4879)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5006..5461
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5093..5112)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5303..5324
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5487..5591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5592..5660,5661..6452)
                     /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(5810..5829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6623..7211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7112..7131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
       61 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
      121 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
      181 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
      241 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
      301 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
      361 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
      421 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
      481 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
      541 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
      601 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
      661 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
      721 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
      781 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
      841 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
      901 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
      961 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     1021 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     1081 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     1141 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     1201 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     1261 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     1321 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     1381 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     1441 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     1501 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     1561 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     1621 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     1681 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     1741 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     1801 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     1861 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     1921 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     1981 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     2041 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat
     2101 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa
     2161 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     2221 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     2281 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     2341 ctgtccaaat atgagagctg tttaagagct atgctggaaa cagcatagca agtttaaata
     2401 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta
     2461 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2521 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2581 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2641 actttggcgc cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2701 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2761 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2821 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2881 ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg
     2941 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     3001 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     3061 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3121 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3181 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3241 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3301 ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3361 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3421 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3481 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     3541 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     3601 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     3661 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     3721 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     3781 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     3841 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     3901 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     3961 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     4021 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     4081 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     4141 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     4201 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     4261 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     4321 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4381 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     4441 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     4501 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4561 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4621 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     4681 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4741 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4801 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4861 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4921 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4981 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5041 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5101 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5161 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5221 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5281 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5341 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5401 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     5461 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5521 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5581 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5641 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5701 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5761 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5821 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5881 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5941 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6001 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6061 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6121 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6181 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6241 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6301 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6361 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6421 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6481 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6541 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6601 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6661 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6721 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6781 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6841 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6901 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6961 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7021 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7081 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7141 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7201 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7261 ttgctcacat gttctttcct gcgttatccc ctgattctgt g
//
