LOCUS       sequence-363666-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Dnmt3a-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-sgDnmt3a#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(87..106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        278..657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        659..857
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     812..832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             875..1055
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1102..1227
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1724..1957
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2142..2186
                     /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    2484..2601
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2683..2931
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2683..2703
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2854..2873
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2953..3028
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3081..3580
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3116..3139)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3513..3532
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3584..3604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3605..4633
                     /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    4689..5277
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4742..4762)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5160..5171)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5280..5296)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5281..5297)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5806..5986
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6048..6636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6128..6147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6807..7598,7599..7667))
                     /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     7430..7449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7668..7760,1..12))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
       61 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
      121 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
      181 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
      241 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
      301 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      361 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      421 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      481 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      541 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      601 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      661 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      721 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      781 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      841 gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg
      901 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
      961 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     1021 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
     1081 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
     1141 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
     1201 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
     1261 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
     1321 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     1381 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     1441 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     1501 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     1561 caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1621 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1681 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1741 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1801 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1861 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1921 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1981 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     2041 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     2101 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     2161 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     2221 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     2281 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     2341 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     2401 acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga
     2461 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2521 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2581 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta
     2641 gtaccgggcc cgctctagcc ctgcaggaac tgtttaaact gtgagggcct atttcccatg
     2701 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
     2761 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     2821 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     2881 aagtatttcg atttcttggc tttatatatc ttgtggaaag gaccaaacac cgcaggccga
     2941 attgtgtctt gggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     3001 acttgaaaaa gtggcaccga gtcggtgctt ttttgtttgg cgcgcctatg gcttaattaa
     3061 catggcggcc gcattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg
     3121 cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca
     3181 ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc
     3241 ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac
     3301 gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca
     3361 atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg
     3421 ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     3481 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     3541 tgccgcgctg ttctcctctt cctcatctcc gggacccggg atgcccaaga agaagaggaa
     3601 ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag
     3661 tgatgaggtt cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca
     3721 tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa
     3781 ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc
     3841 gcgcggtctg gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg
     3901 tcggtccggg ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat
     3961 ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac
     4021 tgatttcgac caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa
     4081 tctggcattt ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat
     4141 cagggttaaa gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac
     4201 gaaaacgctg gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact
     4261 ggtcgagcga tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg
     4321 ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc
     4381 cctggaaggg atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg
     4441 tcagagatac ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc
     4501 ccgcgctgga gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat
     4561 tgtcatgaac tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga
     4621 agatggcgat tagccatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc
     4681 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     4741 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     4801 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     4861 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     4921 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     4981 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     5041 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     5101 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     5161 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     5221 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     5281 gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca
     5341 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt
     5401 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     5461 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     5521 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca
     5581 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt
     5641 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg
     5701 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc
     5761 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta
     5821 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     5881 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     5941 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa
     6001 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     6061 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6721 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6781 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6841 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6901 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6961 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     7021 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     7081 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7141 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7201 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7261 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7321 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7381 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7441 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7501 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7561 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7621 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7741 atttagaaaa ataaacaaat
//
