LOCUS       sequence-363641-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Cdkn2a-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-sgCdkn2a#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..96)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        268..647
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        649..847
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     802..822
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             865..1045
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1092..1217
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1714..1947
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2132..2176
                     /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    2474..2591
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2673..2921
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2673..2693
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2844..2863
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2943..3018
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3071..3570
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3106..3129)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3503..3522
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3574..3594
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3595..4623
                     /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    4679..5267
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4732..4752)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5150..5161)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5270..5286)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5271..5287)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5796..5976
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6038..6626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6118..6137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6797..7588,7589..7657))
                     /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     7420..7439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7658..7760,1..2))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
       61 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      121 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      181 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      241 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      301 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      361 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      421 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      481 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      541 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      601 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      661 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      721 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      781 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      841 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
      901 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      961 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1021 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1081 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1141 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1201 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1261 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1321 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1381 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1441 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1501 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1561 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1621 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1681 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1741 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1801 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1861 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1921 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1981 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     2041 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     2101 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     2161 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     2221 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     2281 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2341 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2401 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag
     2461 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2521 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2581 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc
     2641 cgctctagcc ctgcaggaac tgtttaaact gtgagggcct atttcccatg attccttcat
     2701 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2761 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2821 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2881 atttcttggc tttatatatc ttgtggaaag gaccaaacac cgcggtgcag attcgaactg
     2941 cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     3001 gtggcaccga gtcggtgctt ttttgtttgg cgcgcctatg gcttaattaa catggcggcc
     3061 gcattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     3121 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     3181 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     3241 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     3301 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     3361 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     3421 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     3481 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     3541 ttctcctctt cctcatctcc gggacccggg atgcccaaga agaagaggaa ggtgtccaat
     3601 ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt
     3661 cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa
     3721 atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg
     3781 tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg
     3841 gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg
     3901 ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa
     3961 aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac
     4021 caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt
     4081 ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa
     4141 gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg
     4201 gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga
     4261 tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga
     4321 aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg
     4381 atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac
     4441 ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga
     4501 gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac
     4561 tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat
     4621 tagccatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa
     4681 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     4741 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     4801 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4861 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4921 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4981 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     5041 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     5101 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     5161 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     5221 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     5281 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     5341 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     5401 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5461 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
     5521 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     5581 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     5641 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     5701 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     5761 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     5821 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5881 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5941 tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa
     6001 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6061 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6121 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6181 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6241 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6301 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6361 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6421 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6481 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6541 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6601 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6661 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6721 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6781 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6841 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6901 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6961 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7021 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7081 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7141 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7201 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7261 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7321 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7381 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7441 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7501 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7561 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7621 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7681 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7741 ataaacaaat aggggttccg
//
