LOCUS       sequence-363741-        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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(154..173)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        345..724
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        726..924
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     879..899
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             942..1122
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1169..1294
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1791..2024
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2209..2253
                     /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    2551..2668
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2750..2998
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2750..2770
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2921..2940
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3020..3095
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3148..3647
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3183..3206)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3580..3599
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3651..3671
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3672..4700
                     /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    4756..5344
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4809..4829)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5227..5238)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5347..5363)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5348..5364)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5873..6053
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6115..6703)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6195..6214)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6874..7665,7666..7734))
                     /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     7497..7516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7735..7760,1..79))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
       61 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
      121 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      181 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
      241 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      301 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      361 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      421 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      481 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      541 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      601 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      661 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      721 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      781 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      841 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      901 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
      961 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     1021 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     1081 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     1141 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     1201 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     1261 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     1321 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     1381 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1441 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1501 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1561 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1621 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1681 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1741 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1801 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1861 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1921 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1981 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     2041 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     2101 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     2161 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     2221 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     2281 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     2341 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2401 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     2461 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     2521 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2581 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2641 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     2701 ttggttagta ccgggcccgc tctagccctg caggaactgt ttaaactgtg agggcctatt
     2761 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2821 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2881 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2941 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac caaacaccga
     3001 tgattgaatg ggccctcggg ttttagagct agaaatagca agttaaaata aggctagtcc
     3061 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgtttggcgc gcctatggct
     3121 taattaacat ggcggccgca ttctaccggg taggggaggc gcttttccca aggcagtctg
     3181 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
     3241 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
     3301 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
     3361 gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg
     3421 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
     3481 ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca
     3541 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     3601 gcacgtctgc cgcgctgttc tcctcttcct catctccggg acccgggatg cccaagaaga
     3661 agaggaaggt gtccaattta ctgaccgtac accaaaattt gcctgcatta ccggtcgatg
     3721 caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc caggcgtttt
     3781 ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca tggtgcaagt
     3841 tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat cttctatatc
     3901 ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag ctaaacatgc
     3961 ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc
     4021 ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg
     4081 aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc tgccaggata
     4141 tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata gccgaaattg
     4201 ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta atccatattg
     4261 gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa
     4321 ctaaactggt cgagcgatgg atttccgtct ctggtgtagc tgatgatccg aataactacc
     4381 tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc cagctatcaa
     4441 ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt gatttacggc gctaaggatg
     4501 actctggtca gagatacctg gcctggtctg gacacagtgc ccgtgtcgga gccgcgcgag
     4561 atatggcccg cgctggagtt tcaataccgg agatcatgca agctggtggc tggaccaatg
     4621 taaatattgt catgaactat atccgtaacc tggatagtga aacaggggca atggtgcgcc
     4681 tgctggaaga tggcgattag ccatgtttaa gggttccggt tccactaggt acaattcgat
     4741 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4801 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4861 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     4921 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     4981 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     5041 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     5101 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     5161 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     5221 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     5281 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     5341 ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa tcacaagtag
     5401 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt
     5461 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga
     5521 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     5581 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attggcagaa
     5641 ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct acaagctagt
     5701 accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc gcttgttaca
     5761 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt ggaggtttga
     5821 cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta ctgggtctct
     5881 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     5941 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     6001 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg
     6061 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6121 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6181 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6241 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6301 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6361 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6421 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6481 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6541 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6601 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6661 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6721 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6781 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6841 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6901 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6961 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7021 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7081 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7141 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     7201 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7261 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7321 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7381 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7441 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7501 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7561 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7621 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7681 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7741 cctttttcaa tattattgaa
//
