LOCUS       sequence-182977-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ZWILCH.
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: ZWILCH H12.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    115..136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(151..156,157..174,175..181)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    189..205
                     /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     194..216
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     213..229
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     213..229
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     248..268
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        250..268
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        296..522
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             523..703
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    750..875
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1368..1601
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1786..1830
                     /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        2008..2256
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2008..2028
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2179..2198
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2412..2529
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2598..2809
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2835..3434
                     /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(2835..2854)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3331..3351
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3450..4038
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3503..3523)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3921..3932)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4110..4343
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4421..4542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4458..4477)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4512..4531
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4582..4717
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4737..4756)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4738..4756)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4766..4783)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4766..4782)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4775..4797)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4924..5379
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5011..5030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5221..5242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5405..5509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5510..5578,5579..6370)
                     /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(5728..5747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6541..7129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7030..7049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7283..7300
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
       61 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      121 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      181 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      241 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
      301 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
      361 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
      421 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
      481 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
      541 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      601 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      661 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      721 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      781 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      841 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      901 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      961 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     1021 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1081 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1141 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1201 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1261 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1321 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1381 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1441 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1501 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1561 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1621 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1681 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1741 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1801 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     1861 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     1921 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     1981 cacctcccaa ccccgagggg acccagagag ggcctatttc ccatgattcc ttcatatttg
     2041 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2101 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2161 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2221 ttggctttat atatcttgtg gaaaggacga aacaccgctt tgctgatcaa ctgcactgtt
     2281 taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg
     2341 aaaaagtggc accgagtcgg tgcttttttt gaattctaga tcttgagaca aatggcagta
     2401 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     2461 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     2521 caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg gctcgagtgg
     2581 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2641 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2701 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2761 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg
     2821 cgggatccgc caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     2881 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     2941 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     3001 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     3061 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     3121 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     3181 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
     3241 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     3301 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     3361 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     3421 agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     3481 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3541 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3601 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3661 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3721 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3781 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3841 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3901 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3961 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4021 tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag ctgtagatct
     4081 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     4141 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     4201 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     4261 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     4321 gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta ttattcagta
     4381 tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta
     4441 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4501 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct
     4561 atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4621 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4681 ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct
     4741 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4801 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     4861 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     4921 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4981 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5041 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5101 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     5161 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5221 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     5281 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5341 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     5401 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5521 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5581 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5641 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     5701 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     5761 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     5821 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     5881 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     5941 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6001 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6061 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6121 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6181 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6241 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6301 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6361 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6421 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6481 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6541 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6601 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6661 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     6721 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6781 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6841 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6901 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6961 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7021 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7081 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7141 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     7201 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     7261 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag ga
//
