LOCUS       sequence-182885-        7302 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 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: CENPB C1.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     161..180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     414..431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    548..569
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(584..589,590..607,608..614)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    622..638
                     /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     627..649
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     646..662
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     646..662
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     681..701
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        683..701
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        729..955
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             956..1136
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1183..1308
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1801..2034
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2219..2263
                     /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        2441..2689
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2441..2461
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2612..2631
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2845..2962
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3031..3242
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3268..3867
                     /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(3268..3287)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3764..3784
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3883..4471
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3936..3956)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4354..4365)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4543..4776
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4854..4975
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4891..4910)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4945..4964
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5015..5150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5170..5189)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5171..5189)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5199..5216)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5199..5215)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5208..5230)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5357..5812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5444..5463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5654..5675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5838..5942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5943..6011,6012..6803)
                     /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(6161..6180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6974..7302,1..260)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
       61 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      301 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      361 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      421 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      481 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      541 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      601 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      661 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      721 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      781 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      841 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      901 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      961 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     1021 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     1081 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1141 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1201 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1261 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1321 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1381 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1441 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1501 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1561 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1621 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1681 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1741 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1801 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1861 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1921 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1981 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     2041 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     2101 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     2161 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     2221 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     2281 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     2341 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     2401 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     2461 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2521 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2581 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2641 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg cgtacttgcg
     2701 ctccgacgcc gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc
     2761 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct agatcttgag
     2821 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     2881 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     2941 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg
     3001 ccggctcgag tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     3061 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     3121 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     3181 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     3241 aggtgtcgtg acgcgggatc cgccaccatg accgagtaca agcccacggt gcgcctcgcc
     3301 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     3361 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     3421 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     3481 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     3541 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     3601 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg
     3661 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     3721 gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac
     3781 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     3841 tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac
     3901 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     3961 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4021 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4081 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     4141 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     4201 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     4261 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     4321 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     4381 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     4441 agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg
     4501 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4561 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     4621 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4681 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4741 tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc
     4801 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4861 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4921 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4981 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     5041 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     5101 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     5161 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     5221 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5281 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5341 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5401 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5461 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5521 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5581 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5641 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5701 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5761 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5821 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5881 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5941 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6001 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6061 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6121 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6181 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6241 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6301 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6361 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6421 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6481 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6541 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6601 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6661 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6721 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6781 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6841 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6901 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6961 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7021 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7081 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7141 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7201 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7261 agttaccgga taaggcgcag cggtcgggct gaacgggggg tt
//
