LOCUS       sequence-182880-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CENPE.
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: CENPE C3.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..35
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    152..173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(188..193,194..211,212..218)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    226..242
                     /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     231..253
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     250..266
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     250..266
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     285..305
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        287..305
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        333..559
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             560..740
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    787..912
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1405..1638
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1823..1867
                     /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        2045..2293
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2045..2065
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2216..2235
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2449..2566
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2635..2846
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2872..3471
                     /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(2872..2891)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3368..3388
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3487..4075
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3540..3560)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3958..3969)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4147..4380
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4458..4579
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4495..4514)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4549..4568
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4619..4754
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4774..4793)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4775..4793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4803..4820)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4803..4819)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4812..4834)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4961..5416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5048..5067)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5258..5279
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5442..5546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5547..5615,5616..6407)
                     /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(5765..5784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6578..7166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7067..7086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
       61 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
      121 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      181 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      241 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
      301 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
      361 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
      421 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
      481 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
      541 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
      601 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      661 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      721 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
      781 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
      841 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
      901 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
      961 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     1021 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     1081 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1141 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     1201 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     1261 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     1321 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     1381 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     1441 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     1501 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     1561 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     1621 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     1681 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     1741 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     1801 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     1861 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     1921 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     1981 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     2041 cagagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2101 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2161 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2221 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2281 aggacgaaac accgtggccg tctgcgtgcg agtggtttaa gagctatgct ggaaacagca
     2341 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2401 tttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2461 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2521 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2581 ggacagcaga gatccacttt ggcgccggct cgagtggctc cggtgcccgt cagtgggcag
     2641 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg
     2701 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
     2761 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
     2821 gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg gatccgccac catgaccgag
     2881 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     2941 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3001 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3061 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3121 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3181 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3241 ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3301 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3361 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     3421 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa
     3481 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3541 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3601 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     3661 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     3721 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     3781 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     3841 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     3901 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3961 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4021 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga
     4081 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4141 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4201 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4261 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4321 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4381 gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa
     4441 tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4501 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4561 aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat
     4621 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4681 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4741 cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc
     4801 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4861 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4921 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4981 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5041 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5101 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5161 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5221 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5281 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     5341 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5401 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5461 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5521 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5581 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5641 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5701 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5761 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5821 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5881 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5941 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6001 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6061 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6121 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6181 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6241 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6301 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6361 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6421 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6481 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6721 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7261 accgtattac cgcctttgag tgagctgata ccgctcgccg ca
//
