LOCUS       sequence-182700-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CCNA2.
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: CCNA2 B1.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..38
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    155..176
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(191..196,197..214,215..221)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    229..245
                     /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     234..256
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     253..269
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     253..269
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     288..308
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        290..308
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        336..562
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             563..743
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    790..915
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1408..1641
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1826..1870
                     /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        2048..2296
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2048..2068
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2219..2238
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2452..2569
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2638..2849
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2875..3474
                     /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(2875..2894)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3371..3391
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3490..4078
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3543..3563)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3961..3972)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4150..4383
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4461..4582
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4498..4517)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4552..4571
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4622..4757
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4777..4796)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4778..4796)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4806..4823)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4806..4822)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4815..4837)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4964..5419
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5051..5070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5261..5282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5445..5549
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5550..5618,5619..6410)
                     /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(5768..5787)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6581..7169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7070..7089
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
       61 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      121 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      181 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      241 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      301 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      361 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      421 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      481 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      541 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
      601 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
      661 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
      721 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
      781 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
      841 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
      901 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
      961 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
     1021 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     1081 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     1141 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     1201 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1261 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1321 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1381 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1441 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1501 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1561 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1621 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     1681 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     1741 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     1801 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     1861 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     1921 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     1981 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     2041 acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2101 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2161 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2221 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2281 gaaaggacga aacaccgccg tccaacaacc gcggaccgtt taagagctat gctggaaaca
     2341 gcatagcaag tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     2401 tgcttttttt gaattctaga tcttgagaca aatggcagta ttcatccaca attttaaaag
     2461 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
     2521 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta
     2581 cagggacagc agagatccac tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg
     2641 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     2701 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2761 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2821 ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc caccatgacc
     2881 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     2941 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     3001 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3061 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     3121 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     3181 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     3241 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     3301 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     3361 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     3421 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt
     3481 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     3541 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     3601 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     3661 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     3721 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     3781 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3841 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     3901 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3961 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     4021 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
     4081 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     4141 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     4201 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     4261 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     4321 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     4381 gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     4441 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     4501 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4561 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     4621 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4681 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4741 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     4801 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4861 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     4921 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     4981 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5041 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5101 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5161 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5221 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5281 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5341 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5401 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5461 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5521 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5581 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5641 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5701 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5761 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5821 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5881 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5941 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6001 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6061 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6121 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6181 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6241 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6301 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6361 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6421 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6481 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6541 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6601 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6661 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6721 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6781 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6841 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6901 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6961 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7021 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7081 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7141 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7201 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7261 ataaccgtat taccgccttt gagtgagctg ataccgctcg cc
//
