LOCUS       sequence-182698-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human DBF4.
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: DBF4 C9.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..24
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    141..162
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(177..182,183..200,201..207)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    215..231
                     /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     220..242
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     239..255
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     239..255
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     274..294
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        276..294
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        322..548
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             549..729
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    776..901
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1394..1627
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1812..1856
                     /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        2034..2282
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2034..2054
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2205..2224
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2438..2555
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2624..2835
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2861..3460
                     /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(2861..2880)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3357..3377
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3476..4064
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3529..3549)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3947..3958)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4136..4369
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4447..4568
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4484..4503)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4538..4557
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4608..4743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4763..4782)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4764..4782)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4792..4809)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4792..4808)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4801..4823)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4950..5405
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5037..5056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5247..5268
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5431..5535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5536..5604,5605..6396)
                     /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(5754..5773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6567..7155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7056..7075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
       61 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
      121 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
      181 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
      241 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
      301 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
      361 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
      421 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
      481 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
      541 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
      601 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
      661 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
      721 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
      781 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
      841 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
      901 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
      961 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     1021 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     1081 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1141 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1201 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     1261 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     1321 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     1381 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     1441 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     1501 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     1561 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     1621 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     1681 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     1741 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     1801 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     1861 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     1921 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     1981 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc
     2041 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta
     2101 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     2161 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     2221 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     2281 ccgtccccaa agtggcttac attgtttaag agctatgctg gaaacagcat agcaagttta
     2341 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat
     2401 tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
     2461 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
     2521 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
     2581 atccactttg gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     2641 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
     2701 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     2761 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     2821 gccgccagaa cacaggtgtc gtgacgcggg atccgccacc atgaccgagt acaagcccac
     2881 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     2941 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     3001 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     3061 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     3121 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3181 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3241 cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3301 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3361 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3421 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca
     3481 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3541 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3601 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3661 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3721 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3781 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3841 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     3901 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3961 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     4021 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca
     4081 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     4141 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     4201 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4261 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4321 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt
     4381 agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt
     4441 gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4501 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4561 gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa
     4621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4741 gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     4801 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     4861 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     4921 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     4981 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5041 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5101 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5161 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5221 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5281 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5341 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5401 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5461 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5521 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5581 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5641 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5701 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5761 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5821 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5881 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5941 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6001 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6061 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6121 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6181 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6241 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6301 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6361 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6421 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6481 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6541 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6601 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6661 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6721 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6781 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6841 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6901 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6961 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7021 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7081 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7141 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7201 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7261 gcctttgagt gagctgatac cgctcgccgc agccgaacga cc
//
