LOCUS       sequence-182890-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDC20.
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: CDC20 D4.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..29
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    146..167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(182..187,188..205,206..212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    220..236
                     /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     225..247
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     244..260
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     244..260
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     279..299
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        281..299
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        327..553
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             554..734
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    781..906
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1399..1632
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1817..1861
                     /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        2039..2287
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2039..2059
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2210..2229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2442..2559
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2628..2839
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2865..3464
                     /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(2865..2884)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3361..3381
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3480..4068
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3533..3553)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3951..3962)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4140..4373
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4451..4572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4488..4507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4542..4561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4612..4747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4767..4786)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4768..4786)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4796..4813)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4796..4812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4954..5409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5041..5060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5251..5272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5435..5539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5540..5608,5609..6400)
                     /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(5758..5777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6571..7159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7060..7079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
       61 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
      121 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
      181 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
      241 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
      301 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
      361 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
      421 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
      481 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
      541 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      601 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      661 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
      721 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
      781 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
      841 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
      901 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
      961 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     1021 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1081 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1141 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1201 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     1261 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1321 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1381 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1441 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1501 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1561 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1621 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1681 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     1741 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     1801 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     1861 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     1921 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     1981 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga
     2041 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     2101 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     2161 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     2221 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     2281 aaacaccgcg ctggcagcgc aaagccagtt taagagctat gctggaaaca gcatagcaag
     2341 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     2401 gaattctaga tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     2461 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     2521 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     2581 agagatccac tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac
     2641 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     2701 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2761 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2821 gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc caccatgacc gagtacaagc
     2881 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     2941 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     3001 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     3061 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     3121 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     3181 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     3241 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     3301 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     3361 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     3421 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca
     3481 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     3541 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     3601 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3661 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3721 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3781 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3841 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3901 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3961 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     4021 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag
     4081 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     4141 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     4201 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     4261 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     4321 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta
     4381 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     4441 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4501 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4561 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     4621 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4681 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4741 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4801 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4861 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4921 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4981 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     5041 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5101 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5161 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5221 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5281 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5341 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     5401 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5461 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5521 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5581 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5641 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5701 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5761 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5821 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5881 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5941 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6001 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6061 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6121 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6181 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6241 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6301 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6361 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6421 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6481 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6541 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6601 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6661 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6721 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6781 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6841 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6901 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6961 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7021 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7081 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7141 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7201 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     7261 taccgccttt gagtgagctg ataccgctcg ccgcagccga a
//
