LOCUS       sequence-182705-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CCNB1.
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: CCNB1 B4.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    123..144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(159..164,165..182,183..189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    197..213
                     /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     202..224
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     221..237
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     221..237
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     256..276
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        258..276
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        304..530
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             531..711
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    758..883
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1376..1609
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1794..1838
                     /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        2016..2264
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2016..2036
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2187..2206
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2420..2537
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2606..2817
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2843..3442
                     /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(2843..2862)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3339..3359
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3458..4046
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3511..3531)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3929..3940)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4118..4351
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4429..4550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4466..4485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4520..4539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4590..4725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4745..4764)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4746..4764)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4774..4791)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4774..4790)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4783..4805)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4932..5387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5019..5038)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5229..5250
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5413..5517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5518..5586,5587..6378)
                     /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(5736..5755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6549..7137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7038..7057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7291..7302,1..6)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
       61 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      121 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      181 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      241 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
      301 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      361 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
      421 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
      481 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
      541 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
      601 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
      661 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
      721 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
      781 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      841 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      901 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      961 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     1021 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1081 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1141 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     1201 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1261 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1321 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1381 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1441 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1501 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1561 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1621 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1681 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     1741 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     1801 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     1861 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     1921 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     1981 ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc atgattcctt
     2041 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa
     2101 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     2161 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     2221 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtcttg gcctcagtcc
     2281 gggctgttta agagctatgc tggaaacagc atagcaagtt taaataaggc tagtccgtta
     2341 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga attctagatc ttgagacaaa
     2401 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     2461 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2521 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     2581 tcgagtggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     2641 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     2701 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     2761 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg
     2821 tcgtgacgcg ggatccgcca ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     2881 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     2941 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3001 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3061 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3121 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3181 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     3241 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     3301 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     3361 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     3421 gacccgcaag cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat
     3481 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     3541 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     3601 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     3661 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     3721 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     3781 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     3841 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     3901 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3961 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     4021 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     4081 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4141 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     4201 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     4261 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     4321 cccttttagt cagtgtggaa aatctctagc agtacgtata gtagttcatg tcatcttatt
     4381 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4501 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4561 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4621 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4681 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4741 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4801 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4861 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4921 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4981 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5041 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5101 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5161 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5221 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5281 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5341 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5401 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5461 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5521 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5581 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5641 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5701 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5761 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5821 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5881 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5941 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6001 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6061 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6121 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6181 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6241 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6301 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6361 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6421 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6481 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6541 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6601 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6661 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6721 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6781 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6841 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6901 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6961 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7021 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7081 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7141 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7201 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7261 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tg
//
