LOCUS       sequence-361938-        7760 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 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-sgRasa1#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(91..110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        282..661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        663..861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     816..836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             879..1059
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1106..1231
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1728..1961
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2146..2190
                     /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    2488..2605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..2935
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2687..2707
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2858..2877
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2957..3032
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3085..3584
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3120..3143)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3517..3536
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3588..3608
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3609..4637
                     /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    4693..5281
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4746..4766)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5164..5175)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5284..5300)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5285..5301)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5810..5990
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6052..6640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6132..6151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6811..7602,7603..7671))
                     /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     7434..7453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7672..7760,1..16))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
       61 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      121 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      181 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      241 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      301 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      361 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      421 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      481 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      541 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      601 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      661 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      721 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      781 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      841 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
      901 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      961 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     1021 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1081 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1141 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1201 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1261 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1321 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1381 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1441 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1501 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1561 acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1621 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1681 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1741 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1801 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1861 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1921 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1981 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     2041 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     2101 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     2161 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     2221 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     2281 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     2341 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     2401 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     2461 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2521 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2581 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2641 gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg gcctatttcc
     2701 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     2761 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2821 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2881 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa acaccgttat
     2941 aagagagagt gatcgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     3001 atcaacttga aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc tatggcttaa
     3061 ttaacatggc ggccgcattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3121 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3181 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3241 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3301 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     3361 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     3421 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     3481 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     3541 cgtctgccgc gctgttctcc tcttcctcat ctccgggacc cgggatgccc aagaagaaga
     3601 ggaaggtgtc caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa
     3661 cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg
     3721 agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga
     3781 ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc
     3841 aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc
     3901 atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc
     3961 ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac
     4021 gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac
     4081 gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca
     4141 ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca
     4201 gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta
     4261 aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt
     4321 tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc
     4381 gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact
     4441 ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata
     4501 tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa
     4561 atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc
     4621 tggaagatgg cgattagcca tgtttaaggg ttccggttcc actaggtaca attcgatatc
     4681 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4741 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4801 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     4861 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     4921 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4981 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     5041 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     5101 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     5161 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     5221 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     5281 catcgatacc gtcgacctcg atcgagacct agaaaaacat ggagcaatca caagtagcaa
     5341 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
     5401 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
     5461 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     5521 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
     5581 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
     5641 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
     5701 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
     5761 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
     5821 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     5881 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     5941 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gcatgtgagc
     6001 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6061 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6121 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6181 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6241 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6301 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6361 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6421 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6481 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6541 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6601 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6661 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6721 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6781 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6841 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6901 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6961 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     7021 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7081 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7141 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7201 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7261 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7321 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7381 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7441 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7501 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7561 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7621 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7681 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7741 atgtatttag aaaaataaac
//
