LOCUS       sequence-362046-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Nf2-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-sgNf2#1/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        261..640
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..840
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     795..815
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             858..1038
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1085..1210
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1707..1940
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2125..2169
                     /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    2467..2584
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2666..2914
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2666..2686
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2837..2856
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2935..3010
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3063..3562
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3098..3121)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3495..3514
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3566..3586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3587..4615
                     /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    4671..5259
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4724..4744)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5142..5153)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5262..5278)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5263..5279)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5788..5968
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6030..6618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6110..6129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6789..7580,7581..7649))
                     /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     7412..7431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7650..7754)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
      121 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
      181 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
      241 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      301 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      361 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      421 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      481 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      541 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      601 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      661 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      721 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      781 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      841 cgcgttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      901 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
      961 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     1021 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
     1081 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
     1141 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
     1201 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     1261 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     1321 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1381 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1441 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1501 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg
     1561 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat
     1621 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg
     1681 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca
     1741 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct
     1801 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg
     1861 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac
     1921 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact
     1981 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg
     2041 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt
     2101 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg
     2161 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata
     2221 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat
     2281 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga
     2341 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt
     2401 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat
     2461 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2521 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2581 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttagtaccgg gcccgctcta
     2641 gccctgcagg aactgtttaa actgtgaggg cctatttccc atgattcctt catatttgca
     2701 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     2761 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2821 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2881 ggctttatat atcttgtgga aaggaccaaa caccgcttgg tatgcggagc accggtttta
     2941 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     3001 gagtcggtgc ttttttgttt ggcgcgccta tggcttaatt aacatggcgg ccgcattcta
     3061 ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct
     3121 gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag
     3181 gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt
     3241 caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc
     3301 acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct
     3361 ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag
     3421 gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc
     3481 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc
     3541 ttcctcatct ccgggacccg ggatgcccaa gaagaagagg aaggtgtcca atttactgac
     3601 cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa
     3661 cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct
     3721 gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc
     3781 agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa
     3841 aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg
     3901 accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga
     3961 tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg
     4021 ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat
     4081 tgcttataac accctgttac gtatagccga aattgccagg atcagggtta aagatatctc
     4141 acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac
     4201 cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc
     4261 cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg
     4321 tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga
     4381 agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg
     4441 gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat
     4501 accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg
     4561 taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg attagccatg
     4621 tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc
     4681 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     4741 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     4801 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     4861 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     4921 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     4981 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     5041 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     5101 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     5161 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     5221 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat
     5281 cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat
     5341 tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta
     5401 cctttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     5461 gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc
     5521 taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga
     5581 tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa
     5641 gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat
     5701 gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg
     5761 gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg
     5821 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     5881 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     5941 ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg
     6001 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     6061 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     6121 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     6181 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     6241 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6301 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6361 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6421 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6481 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6541 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6601 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6661 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6721 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6781 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6841 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6901 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6961 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     7021 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     7081 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     7141 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     7201 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     7261 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7321 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7381 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7441 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7501 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7561 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7621 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7681 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7741 ataggggttc cgcgcacat
//
