LOCUS       sequence-182858-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human BUB1.
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: BUB1 A5.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..39
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    156..177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(192..197,198..215,216..222)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    230..246
                     /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     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     254..270
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     254..270
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     289..309
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        291..309
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        337..563
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             564..744
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    791..916
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1409..1642
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1827..1871
                     /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        2049..2297
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2049..2069
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2220..2239
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2453..2570
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2639..2850
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2876..3475
                     /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(2876..2895)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3372..3392
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3491..4079
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3544..3564)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3962..3973)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4151..4384
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4462..4583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4499..4518)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4553..4572
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4623..4758
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4778..4797)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4779..4797)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4807..4824)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4807..4823)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4816..4838)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4965..5420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5052..5071)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5262..5283
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5446..5550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5551..5619,5620..6411)
                     /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(5769..5788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6582..7170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7071..7090
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
       61 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      121 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      181 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      301 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
      361 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
      421 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
      481 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
      541 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
      601 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
      661 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
      721 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
      781 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
      841 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
      901 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
      961 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     1021 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     1081 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     1141 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     1201 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     1261 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1321 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1381 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1441 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1501 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1561 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1621 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     1681 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     1741 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     1801 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     1861 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     1921 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1981 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     2041 gacccagaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     2101 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2161 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2221 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2281 ggaaaggacg aaacaccgcc cgcccaggca atgtacaggt ttaagagcta tgctggaaac
     2341 agcatagcaa gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     2401 gtgctttttt tgaattctag atcttgagac aaatggcagt attcatccac aattttaaaa
     2461 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     2521 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     2581 acagggacag cagagatcca ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg
     2641 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
     2701 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
     2761 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
     2821 tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg ccaccatgac
     2881 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     2941 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3001 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3061 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3121 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3181 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3241 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3301 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3361 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3421 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     3481 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3541 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3601 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3661 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3721 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3781 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3841 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3901 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     3961 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     4021 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     4081 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4141 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     4201 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     4261 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     4321 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     4381 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     4441 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     4501 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4561 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     4621 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4681 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4741 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     4801 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4861 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4921 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     4981 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5041 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5101 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5161 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5221 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5281 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5341 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5401 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     5461 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5521 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5581 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5641 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5701 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5761 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5821 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5881 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5941 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6001 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6061 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6121 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6181 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6241 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     6301 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     6361 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     6421 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6481 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6541 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6601 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6661 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6721 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6781 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6841 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6901 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6961 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7021 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7081 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7141 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7201 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     7261 gataaccgta ttaccgcctt tgagtgagct gataccgctc gc
//
