LOCUS       sequence-180655-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a p53-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  AUTHORS   Rogers ZN, McFarland CD, Winters IP, Naranjo S, Chuang CH, Petrov D,
            Winslow MM
  TITLE     A quantitative and multiplexed approach to uncover the fitness 
            landscape of tumor suppression in vivo.
  JOURNAL   Nat Methods. 2017 May 22. doi: 10.1038/nmeth.4297.
  PUBMED    28530655
REFERENCE   2  (bases 1 to 7760)
  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"; journalName: "Nat 
            Methods. 2017 May 22. doi: 10.1038/nmeth.4297."
COMMENT     Sequence Label: Lenti-sgp53/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        171..550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        552..750
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     705..725
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             768..948
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    995..1120
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1617..1850
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2035..2079
                     /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"
     misc_feature    2377..2494
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2576..2824
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2576..2596
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2747..2766
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2846..2921
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2974..3473
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3009..3032)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3406..3425
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3477..3497
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3498..4526
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    4582..5170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4635..4655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5053..5064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5173..5189)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5174..5190)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5699..5879
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5941..6529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6021..6040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6700..7491,7492..7560))
                     /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     7323..7342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7561..7665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7740..7759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
       61 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
      121 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      181 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      241 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      301 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      361 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      421 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      481 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      541 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      601 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      661 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      721 gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt
      781 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      841 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      901 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      961 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     1021 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     1081 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     1141 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     1201 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     1261 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     1321 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     1381 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     1441 gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg
     1501 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1561 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1621 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1681 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1741 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1801 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1861 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1921 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1981 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     2041 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     2101 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     2161 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     2221 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     2281 ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg
     2341 ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     2401 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     2461 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     2521 tccagtttgg ttagtaccgg gcccgctcta gccctgcagg aactgtttaa actgtgaggg
     2581 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2641 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2701 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2761 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggaccaaa
     2821 caccgaggag ctcctgacac tcggagtttt agagctagaa atagcaagtt aaaataaggc
     2881 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt tggcgcgcct
     2941 atggcttaat taacatggcg gccgcattct accgggtagg ggaggcgctt ttcccaaggc
     3001 agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct
     3061 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     3121 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     3181 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     3241 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     3301 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     3361 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     3421 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggaccc gggatgccca
     3481 agaagaagag gaaggtgtcc aatttactga ccgtacacca aaatttgcct gcattaccgg
     3541 tcgatgcaac gagtgatgag gttcgcaaga acctgatgga catgttcagg gatcgccagg
     3601 cgttttctga gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt
     3661 gcaagttgaa taaccggaaa tggtttcccg cagaacctga agatgttcgc gattatcttc
     3721 tatatcttca ggcgcgcggt ctggcagtaa aaactatcca gcaacatttg ggccagctaa
     3781 acatgcttca tcgtcggtcc gggctgccac gaccaagtga cagcaatgct gtttcactgg
     3841 ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa caggctctag
     3901 cgttcgaacg cactgatttc gaccaggttc gttcactcat ggaaaatagc gatcgctgcc
     3961 aggatatacg taatctggca tttctgggga ttgcttataa caccctgtta cgtatagccg
     4021 aaattgccag gatcagggtt aaagatatct cacgtactga cggtgggaga atgttaatcc
     4081 atattggcag aacgaaaacg ctggttagca ccgcaggtgt agagaaggca cttagcctgg
     4141 gggtaactaa actggtcgag cgatggattt ccgtctctgg tgtagctgat gatccgaata
     4201 actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc accagccagc
     4261 tatcaactcg cgccctggaa gggatttttg aagcaactca tcgattgatt tacggcgcta
     4321 aggatgactc tggtcagaga tacctggcct ggtctggaca cagtgcccgt gtcggagccg
     4381 cgcgagatat ggcccgcgct ggagtttcaa taccggagat catgcaagct ggtggctgga
     4441 ccaatgtaaa tattgtcatg aactatatcc gtaacctgga tagtgaaaca ggggcaatgg
     4501 tgcgcctgct ggaagatggc gattagccat gtttaagggt tccggttcca ctaggtacaa
     4561 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     4621 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4681 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4741 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4801 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4861 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4921 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     4981 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5041 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5101 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5161 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     5221 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     5281 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     5341 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5401 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     5461 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     5521 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     5581 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     5641 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     5701 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5761 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5821 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     5881 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5941 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6001 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6061 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6121 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6181 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6241 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6301 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6361 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6421 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6481 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6541 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6601 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6661 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6721 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6781 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6841 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6901 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6961 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7021 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7081 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7141 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7201 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7261 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7321 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7381 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7441 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7501 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7561 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7621 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7681 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     7741 acaatctgct ctgatgccgc
//
