LOCUS       sequence-182891-        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 D3.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..33
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    150..171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(186..191,192..209,210..216)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    224..240
                     /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     229..251
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     248..264
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     248..264
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     283..303
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        285..303
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        331..557
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             558..738
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    785..910
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1403..1636
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1821..1865
                     /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        2043..2291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2043..2063
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2214..2233
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2632..2843
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2869..3468
                     /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(2869..2888)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3365..3385
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3484..4072
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3537..3557)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3955..3966)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4144..4377
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4455..4576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4492..4511)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4546..4565
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4616..4751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4771..4790)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4772..4790)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4800..4817)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4800..4816)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4809..4831)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4958..5413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5045..5064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5255..5276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5439..5543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5544..5612,5613..6404)
                     /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(5762..5781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6575..7163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7064..7083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
       61 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      121 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      181 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      241 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      301 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
      361 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
      421 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
      481 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
      541 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      601 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
      661 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
      721 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
      781 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
      841 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
      901 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
      961 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     1021 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1081 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1141 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1201 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     1261 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1321 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1381 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1441 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1501 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1561 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1621 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     1681 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     1741 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     1801 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     1861 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     1921 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     1981 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca
     2041 gagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2101 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2161 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2221 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2281 gacgaaacac cgctttgcgc tgccagcgcg cgtttaagag ctatgctgga aacagcatag
     2341 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2401 ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccactttggc gccggctcga gtggctccgg tgcccgtcag tgggcagagc
     2641 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     2701 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     2761 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     2821 acgggtttgc cgccagaaca caggtgtcgt gacgcgggat ccgccaccat gaccgagtac
     2881 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     2941 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     3001 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     3061 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     3121 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     3181 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     3241 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     3301 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     3361 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     3421 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     3481 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     3541 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3601 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3661 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3721 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3781 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3841 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     3901 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     3961 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4021 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     4081 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     4141 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     4201 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     4261 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     4321 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     4381 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     4441 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4501 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4561 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     4621 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4681 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4741 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     4801 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     4861 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     4921 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     4981 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     5041 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5101 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5161 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5221 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5281 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     5341 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5401 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5461 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5521 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5581 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5641 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5701 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5761 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5821 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5881 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5941 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6001 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6061 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6121 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6181 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6241 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6301 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6361 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6421 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6481 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6541 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6601 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6661 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6721 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6781 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6841 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6901 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6961 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7021 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7081 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7141 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7201 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7261 gtattaccgc ctttgagtga gctgataccg ctcgccgcag c
//
