LOCUS       sequence-183038-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human VCP.
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: VCP F11.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..52
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    169..190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(205..210,211..228,229..235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    243..259
                     /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     248..270
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     267..283
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     267..283
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     302..322
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        304..322
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        350..576
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             577..757
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    804..929
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1422..1655
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1840..1884
                     /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        2062..2310
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2062..2082
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2233..2252
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2466..2583
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2652..2863
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2889..3488
                     /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(2889..2908)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3385..3405
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3504..4092
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3557..3577)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3975..3986)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4164..4397
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4475..4596
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4512..4531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4566..4585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4636..4771
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4791..4810)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4792..4810)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4820..4837)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4820..4836)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4829..4851)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4978..5433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5065..5084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5275..5296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5459..5563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5564..5632,5633..6424)
                     /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(5782..5801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6595..7183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7084..7103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
       61 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      121 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      181 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      241 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      301 cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat
      361 gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga
      421 gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta
      481 ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga
      541 gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc
      601 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      661 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      721 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      781 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      841 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      901 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      961 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
     1021 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
     1081 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
     1141 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
     1201 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
     1261 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     1321 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1381 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1441 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1501 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1561 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1621 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1681 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1741 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1801 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1861 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1921 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1981 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     2041 ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata tttgcatata
     2101 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2161 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2221 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2281 ttatatatct tgtggaaagg acgaaacacc gtggaagcgt atcgacccat cgtttaagag
     2341 ctatgctgga aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag
     2401 tggcaccgag tcggtgcttt ttttgaattc tagatcttga gacaaatggc agtattcatc
     2461 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2521 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2581 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gtggctccgg
     2641 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2701 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2761 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2821 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt gacgcgggat
     2881 ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     2941 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     3001 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     3061 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     3121 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     3181 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     3241 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg
     3301 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     3361 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     3421 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     3481 gtgcctgaac gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
     3541 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     3601 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     3661 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     3721 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     3781 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     3841 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     3901 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     3961 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     4021 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     4081 ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca
     4141 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     4201 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     4261 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4321 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4381 gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata
     4441 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     4501 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4561 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     4621 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4681 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4741 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     4801 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     4861 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     4921 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     4981 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5041 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5101 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5161 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5221 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5281 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5341 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5401 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5461 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5521 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6721 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7261 ccctgattct gtggataacc gtattaccgc ctttgagtga gc
//
