LOCUS       sequence-182867-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human BUB1B.
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: BUB1B A7.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    56..77
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(92..97,98..115,116..122)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    130..146
                     /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     135..157
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     154..170
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     154..170
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     189..209
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        191..209
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        237..463
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             464..644
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    691..816
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1309..1542
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1727..1771
                     /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        1949..2197
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1949..1969
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2120..2139
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2353..2470
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2539..2750
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2776..3375
                     /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(2776..2795)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3272..3292
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3391..3979
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3444..3464)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3862..3873)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4051..4284
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4362..4483
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4399..4418)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4453..4472
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4523..4658
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4678..4697)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4679..4697)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4707..4724)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4707..4723)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4716..4738)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4865..5320
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4952..4971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5162..5183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5346..5450
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5451..5519,5520..6311)
                     /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(5669..5688)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6482..7070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6971..6990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7224..7241
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
       61 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      121 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      181 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
      241 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
      301 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
      361 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
      421 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
      481 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
      541 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
      601 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
      661 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
      721 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
      781 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
      841 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
      901 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
      961 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1021 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1081 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1141 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1201 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1261 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1321 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1381 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1441 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1501 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     1561 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     1621 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     1681 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     1741 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     1801 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     1861 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     1921 ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt
     1981 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     2041 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2101 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2161 cttggcttta tatatcttgt ggaaaggacg aaacaccgcg gatcatgtcc acgcttcagt
     2221 ttaagagcta tgctggaaac agcatagcaa gtttaaataa ggctagtccg ttatcaactt
     2281 gaaaaagtgg caccgagtcg gtgctttttt tgaattctag atcttgagac aaatggcagt
     2341 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2401 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2461 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgagtg
     2521 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     2581 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2641 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2701 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac
     2761 gcgggatccg ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     2821 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     2881 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     2941 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3001 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3061 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3121 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     3181 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     3241 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     3301 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     3361 aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa
     3421 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3481 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3541 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3601 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3661 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3721 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3781 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3841 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3901 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3961 ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc
     4021 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     4081 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4141 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     4201 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4261 agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt
     4321 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     4381 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4441 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     4501 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4561 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4621 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     4681 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     4741 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4801 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4861 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4921 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4981 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5041 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5101 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5161 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5221 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5281 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5341 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5401 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5461 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5521 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5581 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5641 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5701 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5761 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5821 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     5881 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     5941 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6001 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6061 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6121 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6181 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6241 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6301 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6361 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6421 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6481 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6541 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6601 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6661 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6721 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6781 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6841 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     6901 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6961 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7021 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7081 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7141 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7201 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     7261 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta at
//
