LOCUS       sequence-183911-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human RACGAP1.
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: RACGAP1 E8.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    129..150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(165..170,171..188,189..195)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    203..219
                     /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     208..230
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     227..243
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     227..243
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     262..282
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        264..282
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        310..536
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             537..717
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    764..889
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1382..1615
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1800..1844
                     /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        2022..2270
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2022..2042
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2193..2212
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2426..2543
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2612..2823
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2849..3448
                     /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(2849..2868)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3345..3365
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3464..4052
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3517..3537)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3935..3946)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4124..4357
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4435..4556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4472..4491)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4526..4545
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4596..4731
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4751..4770)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4752..4770)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4780..4797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4780..4796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4789..4811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4938..5393
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5025..5044)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5235..5256
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5419..5523
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5524..5592,5593..6384)
                     /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(5742..5761)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6555..7143
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7044..7063
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7297..7302,1..12)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
       61 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
      121 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
      181 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
      241 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg
      301 caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt
      361 agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt
      421 ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca
      481 ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt
      541 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      601 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      661 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
      721 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
      781 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
      841 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
      901 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
      961 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
     1021 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     1081 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     1141 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     1201 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
     1261 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     1321 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     1381 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     1441 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     1501 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     1561 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     1621 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1681 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     1741 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     1801 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     1861 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     1921 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     1981 tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga
     2041 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
     2101 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2161 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2221 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gttgaggatt
     2281 tccgtaaaaa ggtttaagag ctatgctgga aacagcatag caagtttaaa taaggctagt
     2341 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc tagatcttga
     2401 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     2461 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     2521 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     2581 gccggctcga gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     2641 gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta
     2701 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     2761 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     2821 caggtgtcgt gacgcgggat ccgccaccat gaccgagtac aagcccacgg tgcgcctcgc
     2881 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     2941 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3001 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3061 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3121 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3181 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     3241 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3301 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     3361 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3421 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     3481 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3541 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3601 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3661 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3721 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3781 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3841 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     3901 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3961 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     4021 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     4081 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     4141 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     4201 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     4261 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4321 ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat
     4381 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     4441 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4501 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4561 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4621 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4681 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     4741 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     4801 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     4861 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     4921 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4981 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5041 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5101 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5161 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5221 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5281 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5341 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     5401 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5461 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5521 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5581 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5641 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5701 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5761 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5821 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5881 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5941 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6001 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6061 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6121 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6181 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6241 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6301 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6361 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6421 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6481 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6541 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6601 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6661 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6721 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6781 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6841 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6901 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6961 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7021 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7081 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7141 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7201 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7261 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg ag
//
