LOCUS       sequence-361942-        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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(124..143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        315..694
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        696..894
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     849..869
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             912..1092
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1139..1264
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1761..1994
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2179..2223
                     /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    2521..2638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2720..2968
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2720..2740
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2891..2910
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2990..3065
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3118..3617
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3153..3176)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3550..3569
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3621..3641
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3642..4670
                     /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    4726..5314
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4779..4799)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5197..5208)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5317..5333)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5318..5334)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5843..6023
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6085..6673)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6165..6184)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6844..7635,7636..7704))
                     /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     7467..7486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7705..7760,1..49))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
       61 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
      121 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
      181 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
      241 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      301 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      361 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      421 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      481 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      541 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      601 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      661 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      721 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      781 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      841 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt
      901 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
      961 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     1021 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     1081 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     1141 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     1201 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     1261 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     1321 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     1381 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     1441 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     1501 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     1561 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc
     1621 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1681 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1741 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1801 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1861 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1921 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1981 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     2041 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     2101 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     2161 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     2221 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     2281 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     2341 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     2401 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2461 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa
     2521 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2581 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2641 ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagccctg
     2701 caggaactgt ttaaactgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2761 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2821 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2881 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2941 atatatcttg tggaaaggac caaacaccga aagaatggcc gagccttggg ttttagagct
     3001 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     3061 ggtgcttttt tgtttggcgc gcctatggct taattaacat ggcggccgca ttctaccggg
     3121 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
     3181 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
     3241 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
     3301 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
     3361 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
     3421 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
     3481 ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg
     3541 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
     3601 catctccggg acccgggatg cccaagaaga agaggaaggt gtccaattta ctgaccgtac
     3661 accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga
     3721 tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg
     3781 tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac
     3841 ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta
     3901 tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa
     3961 gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg
     4021 gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac
     4081 tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt
     4141 ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta
     4201 ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag
     4261 gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct
     4321 ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg
     4381 ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa
     4441 ctcatcgatt gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg
     4501 gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg
     4561 agatcatgca agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc
     4621 tggatagtga aacaggggca atggtgcgcc tgctggaaga tggcgattag ccatgtttaa
     4681 gggttccggt tccactaggt acaattcgat atcaagctta tcgataatca acctctggat
     4741 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     4801 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     4861 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     4921 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     4981 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     5041 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     5101 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     5161 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     5221 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     5281 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga
     5341 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc
     5401 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt
     5461 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     5521 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca
     5581 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc
     5641 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc
     5701 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc
     5761 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg
     5821 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct
     5881 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     5941 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6001 gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6061 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6121 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6181 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6241 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6301 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6361 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6421 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6481 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6541 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6601 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6661 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6721 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6781 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6841 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6901 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6961 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7021 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7081 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7141 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7201 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7261 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7321 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7381 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7441 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7501 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7561 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7621 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7681 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7741 gggttattgt ctcatgagcg
//
