LOCUS       sequence-361940-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Rasa1-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-sgRasa1#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(131..150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        322..701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        703..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     856..876
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             919..1099
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1146..1271
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1768..2001
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2186..2230
                     /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    2528..2645
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2727..2975
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2727..2747
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2898..2917
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2996..3071
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3124..3623
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3159..3182)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3556..3575
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3627..3647
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3648..4676
                     /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    4732..5320
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4785..4805)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5203..5214)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5323..5339)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5324..5340)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5849..6029
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6091..6679)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6171..6190)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6850..7641,7642..7710))
                     /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     7473..7492
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7711..7759,1..56))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
       61 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
      121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
      181 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      241 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      301 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      361 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      421 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      481 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      541 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      601 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      661 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      721 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      781 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      841 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      901 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      961 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     1021 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     1081 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     1141 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     1201 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     1261 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     1321 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     1381 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     1441 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     1501 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     1561 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct
     1621 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1681 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1741 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     1801 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     1861 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     1921 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     1981 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     2041 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     2101 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     2161 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     2221 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     2281 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     2341 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     2401 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     2461 tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca
     2521 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2581 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2641 attttcgggt ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct
     2701 agccctgcag gaactgttta aactgtgagg gcctatttcc catgattcct tcatatttgc
     2761 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     2821 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2881 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2941 tggctttata tatcttgtgg aaaggaccaa acaccgtgcc gggtcaagtt acccggtttt
     3001 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     3061 cgagtcggtg cttttttgtt tggcgcgcct atggcttaat taacatggcg gccgcattct
     3121 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     3181 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta
     3241 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag
     3301 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag
     3361 cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc
     3421 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa
     3481 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt
     3541 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     3601 cttcctcatc tccgggaccc gggatgccca agaagaagag gaaggtgtcc aatttactga
     3661 ccgtacacca aaatttgcct gcattaccgg tcgatgcaac gagtgatgag gttcgcaaga
     3721 acctgatgga catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc
     3781 tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg
     3841 cagaacctga agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa
     3901 aaactatcca gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac
     3961 gaccaagtga cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg
     4021 atgccggtga acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc
     4081 gttcactcat ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga
     4141 ttgcttataa caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatatct
     4201 cacgtactga cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca
     4261 ccgcaggtgt agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt
     4321 ccgtctctgg tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg
     4381 gtgttgccgc gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg
     4441 aagcaactca tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct
     4501 ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa
     4561 taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc
     4621 gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gattagccat
     4681 gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct
     4741 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4801 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4861 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4921 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4981 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     5041 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     5101 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     5161 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     5221 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     5281 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga
     5341 tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga
     5401 ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt
     5461 acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     5521 ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat
     5581 ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc cagggatcag
     5641 atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga
     5701 agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga
     5761 tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat
     5821 ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg
     5881 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
     5941 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
     6001 cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag
     6061 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6121 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6181 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6241 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6301 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6361 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6421 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6481 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6541 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6601 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6661 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6721 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6781 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6841 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6901 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6961 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7021 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7081 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7141 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7201 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7261 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7321 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7381 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7441 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7501 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7561 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7621 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7681 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7741 ttatcagggt tattgtctc
//
