LOCUS       sequence-183357-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CDCA3.
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: CDCA3 B12.6 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    117..138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(153..158,159..176,177..183)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    191..207
                     /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     196..218
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     215..231
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     215..231
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     250..270
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        252..270
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        298..524
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             525..705
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    752..877
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1370..1603
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1788..1832
                     /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        2010..2258
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2010..2030
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2181..2200
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2413..2530
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2599..2810
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2836..3435
                     /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(2836..2855)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3332..3352
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3451..4039
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3504..3524)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3922..3933)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4111..4344
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4422..4543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4459..4478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4513..4532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4583..4718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4738..4757)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4739..4757)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4767..4784)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4767..4783)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4776..4798)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4925..5380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5012..5031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5222..5243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5406..5510
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5511..5579,5580..6371)
                     /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(5729..5748)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6542..7130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7031..7050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7284..7301
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
       61 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      121 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      181 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      241 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt
      301 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct
      361 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg
      421 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
      481 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
      541 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
      601 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
      661 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
      721 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
      781 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
      841 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
      901 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
      961 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     1021 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     1081 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     1141 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     1201 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     1261 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     1321 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     1381 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     1441 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     1501 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     1561 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     1621 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     1681 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     1741 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     1801 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     1861 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     1921 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     1981 cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt ccttcatatt
     2041 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     2101 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2161 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2221 tcttggcttt atatatcttg tggaaaggac gaaacaccgg agggtcttaa acatgcccgt
     2281 ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt
     2341 gaaaaagtgg caccgagtcg gtgctttttt tgaattctag atcttgagac aaatggcagt
     2401 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2461 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2521 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg
     2581 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2641 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2701 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2761 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac
     2821 gcgggatccg ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     2881 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     2941 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     3001 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3061 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3121 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3181 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     3241 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3301 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3361 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     3421 aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa
     3481 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3541 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3601 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3661 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3721 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3781 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3841 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3901 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3961 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4021 ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc
     4081 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     4141 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4201 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     4261 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4321 agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt
     4381 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     4441 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4501 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     4561 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4621 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4681 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     4741 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     4801 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4861 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4921 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4981 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5041 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5101 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5161 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5221 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5281 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5341 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5401 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5461 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5521 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5581 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5641 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5701 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5761 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5821 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5881 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5941 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6001 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6061 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6121 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6181 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6241 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6301 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6361 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6421 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6481 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6541 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6601 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6661 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6721 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6781 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6841 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6901 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6961 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7021 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7081 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7141 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7201 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7261 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga g
//
