LOCUS       sequence-361628-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Kmt2d-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  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 7759)
  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-sgKmt2d#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(168..187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        359..738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        740..938
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     893..913
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             956..1136
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1183..1308
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1805..2038
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2223..2267
                     /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    2565..2682
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2764..3012
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2764..2784
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2935..2954
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3033..3108
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3161..3660
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3196..3219)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3593..3612
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3664..3684
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3685..4713
                     /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    4769..5357
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4822..4842)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5240..5251)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5360..5376)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5361..5377)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5886..6066
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6128..6716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6208..6227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6887..7678,7679..7747))
                     /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     7510..7529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7748..7759,1..93))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
       61 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
      121 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
      181 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      241 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      301 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      361 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      421 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      481 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      541 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      601 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      661 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      721 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      781 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      841 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      901 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      961 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     1021 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     1081 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1141 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1201 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1261 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1321 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1381 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1441 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1501 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1561 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1621 aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag
     1681 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1741 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1801 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1861 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1921 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1981 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     2041 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     2101 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2161 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2221 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2281 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2341 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2401 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2461 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2521 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2581 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2641 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2701 agcagagatc cagtttggtt agtaccgggc ccgctctagc cctgcaggaa ctgtttaaac
     2761 tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2821 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2881 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2941 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     3001 ggaccaaaca ccgagcatcc ggcgcatcaa tggttttaga gctagaaata gcaagttaaa
     3061 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttgg
     3121 cgcgcctatg gcttaattaa catggcggcc gcattctacc gggtagggga ggcgcttttc
     3181 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     3241 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     3301 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     3361 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     3421 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     3481 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     3541 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     3601 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggacccggg
     3661 atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca
     3721 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     3781 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     3841 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     3901 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     3961 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     4021 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     4081 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     4141 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     4201 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     4261 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     4321 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     4381 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     4441 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     4501 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     4561 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     4621 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     4681 gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc ggttccacta
     4741 ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     4801 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4861 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4921 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4981 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     5041 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     5101 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     5161 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     5221 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     5281 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     5341 ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag
     5401 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     5461 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     5521 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     5581 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     5641 ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt
     5701 gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca
     5761 cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag
     5821 agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact
     5881 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     5941 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6001 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6061 ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6121 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6181 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6241 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6301 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6361 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6421 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6481 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6541 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6601 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6661 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6721 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6781 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6841 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6901 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6961 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7021 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7081 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7141 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7201 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7261 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7321 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7381 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7441 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7501 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7561 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7621 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7681 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7741 tactcatact cttcctttt
//
