LOCUS       sequence-182878-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CENPB.
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: CENPB C2.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    125..146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(161..166,167..184,185..191)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    199..215
                     /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     204..226
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     223..239
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     223..239
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     258..278
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        260..278
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        306..532
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             533..713
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    760..885
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1378..1611
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1796..1840
                     /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        2018..2266
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2018..2038
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2189..2208
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2421..2538
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2607..2818
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2844..3443
                     /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(2844..2863)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3340..3360
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3459..4047
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3512..3532)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3930..3941)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4119..4352
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4430..4551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4467..4486)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4521..4540
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4591..4726
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4746..4765)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4747..4765)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4775..4792)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4775..4791)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4784..4806)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4933..5388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5020..5039)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5230..5251
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5414..5518
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5519..5587,5588..6379)
                     /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(5737..5756)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6550..7138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7039..7058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7292..7301,1..8)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
       61 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
      121 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
      181 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
      241 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag
      301 cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
      361 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      421 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      481 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      541 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      601 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      661 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      721 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      781 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      841 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      901 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      961 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
     1021 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
     1081 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
     1141 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
     1201 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
     1261 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1321 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1381 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1441 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1501 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1561 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1621 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1681 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1741 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1801 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1861 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1921 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1981 gtttcagacc cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc
     2041 ttcatatttg catatacgat acaaggctgt tagagagata attagaatta atttgactgt
     2101 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2161 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2221 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcgc gatctcgccc
     2281 ttgcgcgttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
     2341 atcaacttga aaaagtggca ccgagtcggt gctttttttg aattctagat cttgagacaa
     2401 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2461 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2521 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     2581 ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2641 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
     2701 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2761 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
     2821 gtcgtgacgc gggatccgcc accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2881 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2941 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     3001 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     3061 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     3121 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     3181 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg
     3241 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     3301 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     3361 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     3421 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     3481 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3541 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3601 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3661 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3721 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3781 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3841 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3901 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3961 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4021 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     4081 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     4141 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     4201 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     4261 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     4321 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     4381 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     4441 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4501 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4561 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4621 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4681 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     4741 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     4801 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4861 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4921 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4981 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5041 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5101 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5161 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5221 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5281 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5341 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     5401 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5461 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5521 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5581 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5641 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5701 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5761 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5821 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5881 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5941 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6001 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6061 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6121 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6181 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6241 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6301 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6361 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6421 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6481 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6541 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6601 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6661 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6721 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6781 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6841 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6901 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6961 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7021 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7081 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7141 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7201 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7261 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca g
//
