LOCUS       sequence-183149-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SSRP1.
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: SSRP1 G10.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..102
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    219..240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(255..260,261..278,279..285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    293..309
                     /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     298..320
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     317..333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     317..333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     352..372
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        354..372
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        400..626
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             627..807
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    854..979
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1472..1705
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1890..1934
                     /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        2112..2360
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2112..2132
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2283..2302
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2516..2633
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2702..2913
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2939..3538
                     /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(2939..2958)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3435..3455
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3554..4142
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3607..3627)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4025..4036)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4214..4447
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4525..4646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4562..4581)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4616..4635
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4686..4821
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4841..4860)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4842..4860)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4870..4887)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4870..4886)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4879..4901)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5028..5483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5115..5134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5325..5346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5509..5613
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5614..5682,5683..6474)
                     /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(5832..5851)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6645..7233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7134..7153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
       61 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      181 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      241 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      301 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      361 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      421 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      481 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      541 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      601 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      661 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      721 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      781 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      841 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      901 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      961 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     1021 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1081 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1141 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1201 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1261 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1321 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1381 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1441 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1501 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1561 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1621 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1681 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1741 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1801 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1861 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1921 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1981 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     2041 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     2101 ggggacccag agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2161 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2221 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2281 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2341 tgtggaaagg acgaaacacc gtatctggcg ccgtgttgct cgtttaagag ctatgctgga
     2401 aacagcatag caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     2461 tcggtgcttt ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta
     2521 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     2581 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     2641 attacaggga cagcagagat ccactttggc gccggctcga gtggctccgg tgcccgtcag
     2701 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2761 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2821 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2881 ctttttcgca acgggtttgc cgccagaaca caggtgtcgt gacgcgggat ccgccaccat
     2941 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     3001 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3061 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3121 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     3181 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3241 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3301 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag
     3361 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3421 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3481 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     3541 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3601 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3661 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3721 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3781 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3841 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3901 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3961 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     4021 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     4081 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4141 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     4201 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     4261 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     4321 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     4381 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     4441 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     4501 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     4561 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4621 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     4681 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4741 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4801 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     4861 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4921 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4981 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     5041 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5101 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5161 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5221 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5281 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5341 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5401 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5461 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5521 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5581 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5641 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5701 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5761 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5821 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5881 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5941 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6001 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6061 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6121 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6181 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6241 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6301 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6361 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6421 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6481 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6541 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6601 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6661 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6721 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6781 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6841 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6901 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6961 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7021 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7081 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7141 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7201 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7261 ttcctggcct tttgctggcc ttttgctcac atgttctttc ct
//
