LOCUS       sequence-182883-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CENPJ.
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: CENPJ C8.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..34
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    151..172
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(187..192,193..210,211..217)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    225..241
                     /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     230..252
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     249..265
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     249..265
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     284..304
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        286..304
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        332..558
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             559..739
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    786..911
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1404..1637
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1822..1866
                     /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        2044..2292
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2044..2064
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2215..2234
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2448..2565
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2634..2845
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2871..3470
                     /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(2871..2890)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3367..3387
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3486..4074
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3539..3559)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3957..3968)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4146..4379
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4457..4578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4494..4513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4548..4567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4618..4753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4773..4792)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4774..4792)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4802..4819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4802..4818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4811..4833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4960..5415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5047..5066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5257..5278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5441..5545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5546..5614,5615..6406)
                     /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(5764..5783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6577..7165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7066..7085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
       61 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
      121 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      181 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      241 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      301 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
      361 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
      421 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
      481 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
      541 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      601 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      661 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
      721 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
      781 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
      841 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
      901 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
      961 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     1021 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     1081 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     1141 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     1201 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     1261 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1321 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1381 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1441 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1501 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1561 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1621 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1681 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     1741 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     1801 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     1861 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     1921 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     1981 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     2041 agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2101 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2161 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2221 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     2281 ggacgaaaca ccgaatatga ccccagcccg agagtttaag agctatgctg gaaacagcat
     2341 agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     2401 ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     2461 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2521 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2581 gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga
     2641 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     2701 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     2761 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     2821 caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc atgaccgagt
     2881 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     2941 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3001 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3061 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3121 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3181 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3241 tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3301 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3361 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3421 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag
     3481 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3541 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3601 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3661 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3721 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3781 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3841 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     3901 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3961 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     4021 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac
     4081 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     4141 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     4201 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     4261 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     4321 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     4381 tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat
     4441 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4501 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4561 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc
     4621 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4681 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4741 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     4801 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4861 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4921 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4981 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     5041 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     5101 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     5161 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5221 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5281 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5341 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5401 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5461 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5521 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5581 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5641 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5701 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5761 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5821 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5881 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5941 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6001 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6061 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6121 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6181 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6241 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6301 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6361 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6421 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6481 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6541 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6601 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6661 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6721 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6781 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6841 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6901 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6961 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7021 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7081 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7141 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7201 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7261 ccgtattacc gcctttgagt gagctgatac cgctcgccgc ag
//
