LOCUS       sequence-361627-        7760 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 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-sgKmt2d#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        290..669
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        671..869
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     824..844
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             887..1067
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1114..1239
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1736..1969
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2154..2198
                     /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    2496..2613
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2695..2943
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2695..2715
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2866..2885
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2965..3040
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3093..3592
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3128..3151)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3525..3544
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3596..3616
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3617..4645
                     /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    4701..5289
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4754..4774)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5172..5183)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5292..5308)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5293..5309)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5818..5998
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6060..6648)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6140..6159)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6819..7610,7611..7679))
                     /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     7442..7461
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7680..7760,1..24))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
       61 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
      121 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      181 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      241 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      301 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      361 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      421 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      481 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      541 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      601 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      661 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      721 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      781 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      841 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
      901 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      961 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     1021 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     1081 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     1141 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     1201 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     1261 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     1321 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     1381 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1441 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1501 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1561 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     1621 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1681 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1741 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1801 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1861 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1921 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1981 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     2041 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     2101 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     2161 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     2221 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     2281 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2341 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     2401 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
     2461 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2521 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2581 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2641 ccagtttggt tagtaccggg cccgctctag ccctgcagga actgtttaaa ctgtgagggc
     2701 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2761 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2821 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2881 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggaccaaac
     2941 accgttgtgc tctctgtaac tgcggtttta gagctagaaa tagcaagtta aaataaggct
     3001 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt ggcgcgccta
     3061 tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt tcccaaggca
     3121 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     3181 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     3241 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     3301 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     3361 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     3421 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     3481 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     3541 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg ggatgcccaa
     3601 gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg cattaccggt
     3661 cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc
     3721 gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg
     3781 caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct
     3841 atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa
     3901 catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt
     3961 tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc
     4021 gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca
     4081 ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac gtatagccga
     4141 aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca
     4201 tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg
     4261 ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa
     4321 ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct
     4381 atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa
     4441 ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc
     4501 gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac
     4561 caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt
     4621 gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac taggtacaat
     4681 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4741 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4801 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4861 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4921 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4981 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     5041 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     5101 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     5161 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     5221 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5281 cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca
     5341 agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag
     5401 gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct
     5461 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5521 cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg
     5581 cagaactaca caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag
     5641 ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg
     5701 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg
     5761 tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg
     5821 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5881 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5941 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc
     6001 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6061 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6121 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6181 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6241 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6301 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6361 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6421 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6481 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6541 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6601 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6661 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6721 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6781 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6841 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6901 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6961 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7021 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7081 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7141 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7201 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7261 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7321 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7381 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7441 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7501 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7561 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7621 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7681 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7741 atatttgaat gtatttagaa
//
