LOCUS       sequence-182869-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CENPA.
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: CENPA B11.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    117..138
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(153..158,159..176,177..183)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    191..207
                     /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     196..218
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     215..231
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     215..231
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     250..270
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        252..270
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        298..524
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             525..705
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    752..877
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1370..1603
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1788..1832
                     /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        2010..2258
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2010..2030
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2181..2200
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2414..2531
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2600..2811
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2837..3436
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     XTEXXELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2837..2856)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3333..3353
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3452..4040
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3505..3525)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3923..3934)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4112..4345
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4423..4544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4460..4479)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4514..4533
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4584..4719
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4739..4758)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4740..4758)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4768..4785)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4768..4784)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4777..4799)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4926..5381
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5013..5032)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5223..5244
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5407..5511
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5512..5580,5581..6372)
                     /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(5730..5749)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6543..7131
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7032..7051
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7285..7302
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
       61 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      121 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      181 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      241 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt
      301 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct
      361 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg
      421 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc
      481 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag
      541 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
      601 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
      661 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
      721 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
      781 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
      841 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
      901 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
      961 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     1021 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     1081 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     1141 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     1201 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
     1261 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
     1321 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
     1381 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
     1441 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
     1501 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
     1561 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
     1621 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
     1681 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
     1741 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     1801 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     1861 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     1921 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     1981 cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt ccttcatatt
     2041 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     2101 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2161 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2221 tcttggcttt atatatcttg tggaaaggac gaaacaccgc ctcgggcttt cggctccggg
     2281 tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc gttatcaact
     2341 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga caaatggcag
     2401 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2461 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2521 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc cggctcgagt
     2581 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     2641 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     2701 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     2761 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga
     2821 cgcgggatcc gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     2881 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     2941 caccgtcgat ccggaccgcc acatcgagcg ggkcaccgag stgcragaac tcttcctcac
     3001 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     3061 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     3121 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     3181 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca
     3241 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     3301 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     3361 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     3421 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     3481 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3541 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3601 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3661 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3721 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3781 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3841 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3901 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3961 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     4021 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     4081 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     4141 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     4201 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     4261 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     4321 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     4381 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     4441 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4501 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     4561 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4621 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4681 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     4741 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     4801 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4861 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4921 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4981 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5041 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5101 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5161 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5221 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5281 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5341 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     5401 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5461 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5521 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5581 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5641 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5701 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5761 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5821 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5881 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5941 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6001 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6061 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6121 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6181 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6241 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6301 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6361 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6421 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6481 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6541 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6601 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6661 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6721 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6781 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6841 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6901 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6961 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7021 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7081 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7141 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7201 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     7261 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg ag
//
