LOCUS       sequence-374617-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Cdkn2c-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-sgCdkn2c#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(57..76)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        248..627
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        629..827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     782..802
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             845..1025
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1072..1197
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1694..1927
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2112..2156
                     /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    2454..2571
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2653..2901
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2653..2673
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2824..2843
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2923..2998
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3051..3550
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3086..3109)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3483..3502
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3554..3574
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3575..4603
                     /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    4659..5247
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4712..4732)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5130..5141)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5250..5266)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5251..5267)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5776..5956
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6018..6606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6098..6117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6777..7568,7569..7637))
                     /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     7400..7419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7638..7742)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
       61 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
      121 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
      181 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      241 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      301 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      361 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      421 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      481 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      541 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      601 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      661 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      721 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      781 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg
      841 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      901 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      961 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     1021 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
     1081 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     1141 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     1201 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     1261 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1321 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1381 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1441 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1501 gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg
     1561 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     1621 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     1681 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     1741 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     1801 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     1861 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     1921 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     1981 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     2041 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     2101 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     2161 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     2221 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     2281 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     2341 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     2401 gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa
     2461 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     2521 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     2581 tacagggaca gcagagatcc agtttggtta gtaccgggcc cgctctagcc ctgcaggaac
     2641 tgtttaaact gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2701 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     2761 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2821 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2881 ttgtggaaag gaccaaacac cgtggcgtcc gcagctgcca gggttttaga gctagaaata
     2941 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     3001 ttttgtttgg cgcgcctatg gcttaattaa catggcggcc gcattctacc gggtagggga
     3061 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     3121 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc
     3181 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     3241 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     3301 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     3361 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     3421 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     3481 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     3541 gggacccggg atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa
     3601 tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat
     3661 gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg
     3721 gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga
     3781 tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca
     3841 acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag
     3901 caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg
     3961 tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga
     4021 aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac
     4081 cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg
     4141 tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga
     4201 gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt
     4261 agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc
     4321 atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg
     4381 attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag
     4441 tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat
     4501 gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag
     4561 tgaaacaggg gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc
     4621 ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     4681 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     4741 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     4801 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     4861 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     4921 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     4981 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     5041 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     5101 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     5161 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     5221 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa
     5281 aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta
     5341 gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca
     5401 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     5461 gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa
     5521 ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc
     5581 tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa
     5641 ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
     5701 gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg
     5761 catccggact gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     5821 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     5881 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     5941 tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6601 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6661 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6721 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6841 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6901 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6961 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7021 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7081 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7141 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7201 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7261 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7321 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7381 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7441 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7501 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7561 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7621 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7681 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7741 cgcacatttc cccgaaaagt
//
