LOCUS       sequence-362050-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Keap1-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-sgKeap1#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(138..157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        329..708
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        710..908
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     863..883
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             926..1106
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1153..1278
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1775..2008
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2193..2237
                     /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    2535..2652
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2734..2982
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2734..2754
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2905..2924
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3004..3079
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3132..3631
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3167..3190)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3564..3583
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3635..3655
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3656..4684
                     /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    4740..5328
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4793..4813)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5211..5222)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5331..5347)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5332..5348)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5857..6037
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6099..6687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6179..6198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6858..7649,7650..7718))
                     /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     7481..7500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7719..7760,1..63))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
       61 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
      121 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      181 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      241 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      301 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      361 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      421 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      481 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      541 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      601 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      661 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      721 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      781 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      841 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      901 ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
      961 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     1021 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     1081 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     1141 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     1201 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     1261 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     1321 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     1381 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     1441 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     1501 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     1561 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     1621 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1681 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1741 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1801 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1861 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1921 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1981 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     2041 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     2101 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     2161 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     2221 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     2281 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     2341 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2401 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2461 gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca
     2521 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2581 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2641 attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccgggc
     2701 ccgctctagc cctgcaggaa ctgtttaaac tgtgagggcc tatttcccat gattccttca
     2761 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     2821 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2881 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2941 gatttcttgg ctttatatat cttgtggaaa ggaccaaaca ccgtcaaata cgactgcccg
     3001 caggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     3061 agtggcaccg agtcggtgct tttttgtttg gcgcgcctat ggcttaatta acatggcggc
     3121 cgcattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc
     3181 agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc
     3241 caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc
     3301 tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat
     3361 ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg
     3421 ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag
     3481 gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc
     3541 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
     3601 gttctcctct tcctcatctc cgggacccgg gatgcccaag aagaagagga aggtgtccaa
     3661 tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga gtgatgaggt
     3721 tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc atacctggaa
     3781 aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata accggaaatg
     3841 gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg cgcgcggtct
     3901 ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc gtcggtccgg
     3961 gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga
     4021 aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga
     4081 ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta atctggcatt
     4141 tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga tcagggttaa
     4201 agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa cgaaaacgct
     4261 ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac tggtcgagcg
     4321 atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt gccgggtcag
     4381 aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg ccctggaagg
     4441 gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg gtcagagata
     4501 cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg
     4561 agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa
     4621 ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga
     4681 ttagccatgt ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata
     4741 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4801 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     4861 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     4921 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     4981 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     5041 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5101 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     5161 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5221 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5281 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     5341 gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
     5401 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
     5461 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     5521 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
     5581 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
     5641 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
     5701 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
     5761 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
     5821 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
     5881 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5941 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     6001 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa
     6061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7741 tgaagcattt atcagggtta
//
