LOCUS       sequence-183318-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDCA8.
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: CDCA8 B6.6 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    127..148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(163..168,169..186,187..193)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    201..217
                     /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     206..228
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     225..241
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     225..241
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     260..280
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        262..280
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        308..534
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             535..715
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    762..887
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1380..1613
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1798..1842
                     /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        2020..2268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2020..2040
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2191..2210
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2423..2540
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2609..2820
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2846..3445
                     /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(2846..2865)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3342..3362
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3461..4049
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3514..3534)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3932..3943)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4121..4354
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4432..4553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4469..4488)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4523..4542
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4593..4728
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4748..4767)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4749..4767)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4777..4794)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4777..4793)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4786..4808)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4935..5390
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5022..5041)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5232..5253
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5416..5520
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5521..5589,5590..6381)
                     /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(5739..5758)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6552..7140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7041..7060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7294..7301,1..10)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
       61 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      121 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
      181 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      241 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
      301 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
      361 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      421 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      481 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      541 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      601 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      661 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      721 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      781 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      841 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      901 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      961 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1021 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1081 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1141 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1201 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1261 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1321 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1381 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1441 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1501 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1561 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1621 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1681 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1741 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1801 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1861 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1921 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1981 tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt
     2041 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
     2101 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     2161 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     2221 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc aggatctcga
     2281 tgttgtaggt ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg
     2341 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaattctag atcttgagac
     2401 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     2461 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     2521 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc
     2581 ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     2641 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     2701 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     2761 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     2821 gtgtcgtgac gcgggatccg ccaccatgac cgagtacaag cccacggtgc gcctcgccac
     2881 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     2941 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3001 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3061 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3121 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3181 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     3241 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3301 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3361 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3421 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa
     3481 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3541 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3601 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3661 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3721 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3781 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3841 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     3901 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3961 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4021 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     4081 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4141 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4201 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4261 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4321 agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt
     4381 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     4441 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4501 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4561 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4621 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4681 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     4741 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     4801 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     4861 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     4921 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4981 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5041 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5101 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5161 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5221 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5281 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5341 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5401 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5461 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5521 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5581 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5641 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5701 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5761 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5821 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5881 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5941 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6001 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6061 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6121 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6181 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6241 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6301 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6361 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6421 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6661 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7201 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7261 gataccgctc gccgcagccg aacgaccgag cgcagcgagt c
//
