LOCUS       sequence-363750-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Tet2-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-sgTet2#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2643..2891
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2643..2663
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2814..2833
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2913..2988
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3041..3540
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3076..3099)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3473..3492
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3544..3564
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3565..4593
                     /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    4649..5237
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4702..4722)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5120..5131)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5240..5256)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5241..5257)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5766..5946
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6008..6596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6088..6107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6767..7558,7559..7627))
                     /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     7390..7409
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7628..7732)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagcc ctgcaggaac tgtttaaact
     2641 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2701 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2761 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2821 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2881 gaccaaacac cgcattctga ggagaacaca cggttttaga gctagaaata gcaagttaaa
     2941 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttgg
     3001 cgcgcctatg gcttaattaa catggcggcc gcattctacc gggtagggga ggcgcttttc
     3061 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     3121 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     3181 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     3241 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     3301 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     3361 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     3421 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     3481 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggacccggg
     3541 atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca
     3601 ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat
     3661 cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg
     3721 gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat
     3781 tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc
     3841 cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt
     3901 tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag
     3961 gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat
     4021 cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt
     4081 atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg
     4141 ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt
     4201 agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat
     4261 ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc
     4321 agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac
     4381 ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc
     4441 ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt
     4501 ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg
     4561 gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc ggttccacta
     4621 ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     4681 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4741 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4801 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4861 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     4921 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     4981 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     5041 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     5101 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     5161 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     5221 ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag
     5281 caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag
     5341 aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca
     5401 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     5461 actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc
     5521 ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt
     5581 gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca
     5641 cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag
     5701 agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact
     5761 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     5821 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     5881 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     5941 ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6001 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6061 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6121 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6181 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6241 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6301 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6361 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6421 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6481 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6541 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6601 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6661 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6721 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6781 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6841 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6901 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6961 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7021 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7081 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7141 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7201 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7261 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7321 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7381 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7441 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7501 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7561 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7621 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7681 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7741 cccgaaaagt gccacctgac
//
