LOCUS       sequence-183130-        7302 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 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: TOP2A G11.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    68..89
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(104..109,110..127,128..134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    142..158
                     /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     147..169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     166..182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     166..182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     201..221
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        203..221
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        249..475
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             476..656
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    703..828
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1321..1554
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1739..1783
                     /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        1961..2209
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1961..1981
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2132..2151
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2365..2482
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2551..2762
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2788..3387
                     /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(2788..2807)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3284..3304
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3403..3991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3456..3476)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3874..3885)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4063..4296
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4374..4495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4411..4430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4465..4484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4535..4670
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4690..4709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4691..4709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4719..4736)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4719..4735)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4728..4750)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4877..5332
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4964..4983)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5174..5195
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5358..5462
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5463..5531,5532..6323)
                     /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(5681..5700)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6494..7082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6983..7002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7236..7253
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
       61 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      121 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      181 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      241 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      301 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      361 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      421 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      481 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      541 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      601 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      661 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      721 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      781 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      841 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      901 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      961 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1021 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1081 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1141 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1201 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1261 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1321 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1381 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1441 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1501 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1561 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1621 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1681 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1741 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1801 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1861 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1921 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     1981 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2041 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2101 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2161 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ctccgcccag
     2221 acacctacat gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc
     2281 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct agatcttgag
     2341 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     2401 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     2461 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg
     2521 ccggctcgag tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     2581 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     2641 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     2701 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     2761 aggtgtcgtg acgcgggatc cgccaccatg accgagtaca agcccacggt gcgcctcgcc
     2821 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     2881 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     2941 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3001 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3061 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3121 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     3181 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3241 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3301 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3361 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
     3421 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3481 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     3541 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     3601 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     3661 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     3721 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     3781 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     3841 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     3901 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     3961 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     4021 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4081 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     4141 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4201 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4261 tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc
     4321 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4381 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4441 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4501 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4561 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4621 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     4681 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4741 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4801 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4861 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4921 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4981 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5041 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5101 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5161 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5221 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5281 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5341 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5401 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5461 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5521 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5581 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5641 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5701 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5761 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5821 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5881 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5941 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6001 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6061 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6121 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6181 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6241 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6301 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6361 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6421 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6481 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6541 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6601 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6661 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6721 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6781 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6841 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6901 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6961 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7021 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7081 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7141 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7201 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7261 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gc
//
