LOCUS       sequence-363622-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Kdm6a-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  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 7748)
  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-sgKdm6a#1/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(101..120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        292..671
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        673..871
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     826..846
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             889..1069
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1116..1241
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1738..1971
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2156..2200
                     /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    2498..2615
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2697..2945
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2697..2717
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2868..2887
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2967..3042
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3095..3582
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3130..3153)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3515..3534
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3586..3606
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3607..4635
                     /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    4691..5279
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4744..4764)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5162..5173)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5282..5298)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5283..5299)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5808..5988
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6050..6638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6130..6149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6809..7600,7601..7669))
                     /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     7432..7451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7670..7748,1..26))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
       61 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
      121 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      181 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      241 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      301 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      361 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      421 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      481 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      541 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      601 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      661 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      721 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      781 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      841 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      901 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      961 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     1021 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1081 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1141 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1201 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1261 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1321 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1381 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1441 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1501 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1561 agaccaccgc acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag
     1621 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1681 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1741 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1801 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1861 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1921 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1981 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     2041 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     2101 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     2161 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     2221 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     2281 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     2341 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     2401 aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc
     2461 gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
     2521 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
     2581 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
     2641 atccagtttg gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg
     2701 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2761 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2821 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2881 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa
     2941 acaccgaatt cgtgctgcaa gtccaggttt tagagctaga aatagcaagt taaaataagg
     3001 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc
     3061 tatggcttaa ttaacatggc ggccgcattc taccgggtag gggaggcgct tttcccaagg
     3121 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     3181 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     3241 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3301 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3361 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     3421 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     3481 ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg
     3541 tctgccgcgc tgttctcctc ttcctcatct ccgggacccg ggatgcccaa gaagaagagg
     3601 aaggtgtcca atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg
     3661 agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag
     3721 catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat
     3781 aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct atatcttcag
     3841 gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat
     3901 cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg
     3961 atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc
     4021 actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt
     4081 aatctggcat ttctggggat tgcttataac accctgttac gtatagccga aattgccagg
     4141 atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga
     4201 acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa
     4261 ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt
     4321 tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc
     4381 gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactct
     4441 ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagatatg
     4501 gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac caatgtaaat
     4561 attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt gcgcctgctg
     4621 gaagatggcg attagccatg tttaagggtt ccggttccac taggtacaat tcgatatcaa
     4681 gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     4741 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     4801 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     4861 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     4921 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     4981 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     5041 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     5101 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     5161 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     5221 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     5281 tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata
     5341 cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt
     5401 ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct gtagatctta
     5461 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     5521 atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca
     5581 caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag
     5641 ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg
     5701 tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc
     5761 gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt
     5821 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     5881 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     5941 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa
     6001 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6061 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6121 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6181 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6241 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6301 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6361 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6421 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6481 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6541 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6601 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6661 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6721 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6781 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6841 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6901 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6961 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7021 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7081 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7141 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7201 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7261 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7321 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7381 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7441 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7501 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7561 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7621 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7681 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7741 gtatttag
//
