LOCUS       sequence-380558-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Tsc1-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-sgTsc1#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(65..84)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        256..635
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        637..835
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     790..810
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             853..1033
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1080..1205
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1702..1935
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2120..2164
                     /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    2462..2579
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2661..2909
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2661..2681
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2832..2851
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2931..3006
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3059..3558
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3094..3117)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3491..3510
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3562..3582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3583..4611
                     /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    4667..5255
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4720..4740)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5138..5149)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5258..5274)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5259..5275)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5784..5964
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6026..6614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6106..6125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6785..7576,7577..7645))
                     /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     7408..7427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7646..7750)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
       61 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
      121 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
      181 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
      241 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
      301 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      361 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      421 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      481 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      541 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      601 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      661 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      721 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      781 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt
      841 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
      901 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
      961 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     1021 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
     1081 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     1141 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     1201 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     1261 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     1321 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     1381 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     1441 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     1501 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccggc cgctgatctt
     1561 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     1621 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     1681 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     1741 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     1801 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     1861 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     1921 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     1981 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     2041 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     2101 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     2161 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     2221 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     2281 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     2341 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     2401 agtgaacgga tcggcactgc gtgcgccaat tctgcagaca aatggcagta ttcatccaca
     2461 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     2521 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     2581 gggtttatta cagggacagc agagatccag tttggttagt accgggcccg ctctagccct
     2641 gcaggaactg tttaaactgt gagggcctat ttcccatgat tccttcatat ttgcatatac
     2701 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2761 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2821 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2881 tatatatctt gtggaaagga ccaaacaccg acctaagatc cacataccag gttttagagc
     2941 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     3001 cggtgctttt ttgtttggcg cgcctatggc ttaattaaca tggcggccgc attctaccgg
     3061 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc
     3121 acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
     3181 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
     3241 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
     3301 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
     3361 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     3421 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     3481 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     3541 tcatctccgg gacccgggat gcccaagaag aagaggaagg tgtccaattt actgaccgta
     3601 caccaaaatt tgcctgcatt accggtcgat gcaacgagtg atgaggttcg caagaacctg
     3661 atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat gcttctgtcc
     3721 gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa
     3781 cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc agtaaaaact
     3841 atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct gccacgacca
     3901 agtgacagca atgctgtttc actggttatg cggcggatcc gaaaagaaaa cgttgatgcc
     3961 ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca ggttcgttca
     4021 ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct ggggattgct
     4081 tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga tatctcacgt
     4141 actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt tagcaccgca
     4201 ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg gatttccgtc
     4261 tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa aaatggtgtt
     4321 gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat ttttgaagca
     4381 actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct ggcctggtct
     4441 ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt ttcaataccg
     4501 gagatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta tatccgtaac
     4561 ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgatta gccatgttta
     4621 agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga
     4681 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     4741 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     4801 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     4861 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     4921 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     4981 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     5041 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     5101 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     5161 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     5221 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag
     5281 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
     5341 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
     5401 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     5461 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
     5521 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
     5581 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
     5641 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
     5701 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
     5761 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
     5821 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     5881 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     5941 agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc
     6001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7081 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7621 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7741 gggttccgcg cacatttccc
//
