LOCUS       sequence-363580-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Tp53-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  AUTHORS   Cai H, Chew SK, Li C, Tsai MK, Andrejka L, Murray CW, Hughes NW, 
            Shuldiner EG, Ashkin EL, Tang R, Hung KL, Chen LC, Lee SYC, Yousefi 
            M, Lin WY, Kunder CA, Cong L, McFarland CD, Petrov DA, Swanton C, 
            Winslow MM
  TITLE     A functional taxonomy of tumor suppression in oncogenic KRAS-driven 
            lung cancer.
  JOURNAL   Cancer Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325.
  PUBMED    33608386
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: "Cancer 
            Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325."
COMMENT     Sequence Label: Lenti-sgTp53#3/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2..106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(181..200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        372..751
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        753..951
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     906..926
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             969..1149
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1196..1321
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1818..2051
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2236..2280
                     /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    2578..2695
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2777..3025
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2777..2797
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2948..2967
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3047..3122
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3175..3674
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3210..3233)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3607..3626
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3678..3698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3699..4727
                     /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    4783..5371
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4836..4856)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5254..5265)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5374..5390)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5375..5391)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5900..6080
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6142..6730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6222..6241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6901..7692,7693..7760,1))
                     /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     7524..7543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
       61 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      121 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
      181 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      241 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
      301 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      361 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      421 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      481 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      541 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      601 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      661 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      721 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      781 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      841 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      901 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg
      961 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     1021 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     1081 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     1141 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     1201 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     1261 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     1321 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     1381 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     1441 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     1501 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1561 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1621 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct gatcttcaga
     1681 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     1741 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     1801 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     1861 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     1921 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     1981 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     2041 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     2101 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     2161 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     2221 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     2281 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     2341 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     2401 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     2461 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     2521 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     2581 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2641 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2701 ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct agccctgcag
     2761 gaactgttta aactgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2821 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2881 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2941 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     3001 tatcttgtgg aaaggaccaa acaccgaaca gatcgtccat gcagtggttt tagagctaga
     3061 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     3121 gcttttttgt ttggcgcgcc tatggcttaa ttaacatggc ggccgcattc taccgggtag
     3181 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     3241 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     3301 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     3361 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     3421 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     3481 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     3541 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     3601 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     3661 ctccgggacc cgggatgccc aagaagaaga ggaaggtgtc caatttactg accgtacacc
     3721 aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg
     3781 acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt
     3841 gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg
     3901 aagatgttcg cgattatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc
     3961 agcaacattt gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg
     4021 acagcaatgc tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg
     4081 aacgtgcaaa acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca
     4141 tggaaaatag cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata
     4201 acaccctgtt acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg
     4261 acggtgggag aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg
     4321 tagagaaggc acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg
     4381 gtgtagctga tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg
     4441 cgccatctgc caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc
     4501 atcgattgat ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac
     4561 acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga
     4621 tcatgcaagc tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg
     4681 atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg cgattagcca tgtttaaggg
     4741 ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac
     4801 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     4861 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     4921 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     4981 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     5041 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     5101 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5161 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5221 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5281 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5341 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg atcgagacct
     5401 agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg
     5461 gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag
     5521 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     5581 ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac
     5641 acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact
     5701 gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa
     5761 tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga
     5821 gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga
     5881 gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     5941 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     6001 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     6061 agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6121 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6181 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6241 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6301 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6361 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6421 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6481 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6541 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6601 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6661 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6721 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6781 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6841 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6901 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6961 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7021 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7081 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7141 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7201 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7261 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7321 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7381 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7441 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7501 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7561 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7621 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7681 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7741 acggaaatgt tgaatactca
//
