LOCUS       sequence-374618-        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#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(78..97)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        269..648
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        650..848
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     803..823
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             866..1046
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1093..1218
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1715..1948
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2133..2177
                     /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    2475..2592
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2674..2922
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2674..2694
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2845..2864
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2944..3019
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3072..3571
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3107..3130)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3504..3523
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3575..3595
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3596..4624
                     /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    4680..5268
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4733..4753)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5151..5162)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5271..5287)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5272..5288)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5797..5977
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6039..6627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6119..6138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6798..7589,7590..7658))
                     /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     7421..7440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7659..7760,1..3))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
       61 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      121 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      181 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      241 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      301 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      361 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      421 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      481 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      541 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      601 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      661 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      721 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      781 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      841 ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga
      901 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
      961 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     1021 ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg
     1081 aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc
     1141 gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga
     1201 gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt
     1261 cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg
     1321 gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa
     1381 atactgggac agctacaacc atcccttcag acaggatcag aagaacttag atcattatat
     1441 aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa
     1501 gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc
     1561 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
     1621 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
     1681 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
     1741 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
     1801 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
     1861 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
     1921 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
     1981 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
     2041 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
     2101 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
     2161 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
     2221 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
     2281 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
     2341 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
     2401 gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca
     2461 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2521 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2581 attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccgggc
     2641 ccgctctagc cctgcaggaa ctgtttaaac tgtgagggcc tatttcccat gattccttca
     2701 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     2761 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2821 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2881 gatttcttgg ctttatatat cttgtggaaa ggaccaaaca ccgatcgtgt ggctcctgca
     2941 agggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     3001 agtggcaccg agtcggtgct tttttgtttg gcgcgcctat ggcttaatta acatggcggc
     3061 cgcattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc
     3121 agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc
     3181 caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc
     3241 tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat
     3301 ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg
     3361 ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag
     3421 gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc
     3481 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
     3541 gttctcctct tcctcatctc cgggacccgg gatgcccaag aagaagagga aggtgtccaa
     3601 tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga gtgatgaggt
     3661 tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc atacctggaa
     3721 aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata accggaaatg
     3781 gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg cgcgcggtct
     3841 ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc gtcggtccgg
     3901 gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga
     3961 aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga
     4021 ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta atctggcatt
     4081 tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga tcagggttaa
     4141 agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa cgaaaacgct
     4201 ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac tggtcgagcg
     4261 atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt gccgggtcag
     4321 aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg ccctggaagg
     4381 gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg gtcagagata
     4441 cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg
     4501 agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa
     4561 ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga
     4621 ttagccatgt ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata
     4681 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4741 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     4801 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     4861 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     4921 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     4981 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5041 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     5101 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5161 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5221 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     5281 gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
     5341 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
     5401 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     5461 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
     5521 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
     5581 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
     5641 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
     5701 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
     5761 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
     5821 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     5881 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     5941 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa
     6001 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6061 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6121 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6181 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6241 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6301 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6361 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6421 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6481 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6541 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6601 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6661 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6721 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6781 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6841 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6901 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6961 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7021 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7081 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7141 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7201 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7261 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7321 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7381 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7441 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7501 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7561 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7621 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7681 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7741 aataaacaaa taggggttcc
//
