LOCUS       sequence-183312-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CENPW.
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: CENPW H3.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    131..152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(167..172,173..190,191..197)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    205..221
                     /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     210..232
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     229..245
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     229..245
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     264..284
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        266..284
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        312..538
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             539..719
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    766..891
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1384..1617
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1802..1846
                     /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        2024..2272
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2024..2044
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2195..2214
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2428..2545
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2614..2825
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2851..3450
                     /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(2851..2870)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3347..3367
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3466..4054
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3519..3539)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3937..3948)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4126..4359
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4437..4558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4474..4493)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4528..4547
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4598..4733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4753..4772)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4754..4772)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4782..4799)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4782..4798)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4791..4813)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4940..5395
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5027..5046)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5237..5258
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5421..5525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5526..5594,5595..6386)
                     /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(5744..5763)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6557..7145
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7046..7065
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7299..7302,1..14)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
       61 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
      121 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
      181 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
      241 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
      301 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
      361 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
      421 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
      481 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
      541 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      601 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      661 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      721 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      781 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      841 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      901 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
      961 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1021 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1081 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1141 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1201 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1261 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1321 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1381 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1441 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1501 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1561 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1621 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1681 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1741 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1801 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1861 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1921 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     1981 attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat
     2041 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     2101 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2161 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2221 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaaagaag
     2281 cctcaacttc gtcgtttaag agctatgctg gaaacagcat agcaagttta aataaggcta
     2341 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat tctagatctt
     2401 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2461 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2521 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2581 gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2641 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2701 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2761 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2821 cacaggtgtc gtgacgcggg atccgccacc atgaccgagt acaagcccac ggtgcgcctc
     2881 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     2941 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3001 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3061 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3121 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3181 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     3241 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     3301 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     3361 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     3421 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     3481 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3541 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3601 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3661 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3721 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3781 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3841 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3901 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3961 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     4021 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
     4081 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     4141 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     4201 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     4261 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     4321 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
     4381 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4441 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4501 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4561 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4621 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4681 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     4741 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     4801 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4861 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4921 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4981 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5041 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5101 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5161 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5221 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5281 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5341 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5401 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5461 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5521 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5581 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5641 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5701 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5761 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5821 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5881 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5941 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6001 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6061 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6121 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6181 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6241 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6301 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6361 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6421 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6481 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6541 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6601 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6661 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6721 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6781 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6841 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6901 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6961 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7021 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7081 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7141 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7201 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     7261 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cg
//
