LOCUS       sequence-363593-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Rnf43-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-sgRnf43#1/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /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        2943..3018
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3071..3570
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3106..3129)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3503..3522
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3574..3594
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3595..4623
                     /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    4679..5267
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4732..4752)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5150..5161)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5270..5286)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5271..5287)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5796..5976
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6038..6626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6118..6137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6797..7588,7589..7657))
                     /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     7420..7439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7658..7759,1..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 ccgtagacga gcccgagccg
     2941 aggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     3001 gtggcaccga gtcggtgctt ttttgtttgg cgcgcctatg gcttaattaa catggcggcc
     3061 gcattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     3121 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     3181 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     3241 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     3301 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     3361 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     3421 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     3481 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     3541 ttctcctctt cctcatctcc gggacccggg atgcccaaga agaagaggaa ggtgtccaat
     3601 ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt
     3661 cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa
     3721 atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg
     3781 tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg
     3841 gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg
     3901 ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa
     3961 aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac
     4021 caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt
     4081 ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa
     4141 gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg
     4201 gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga
     4261 tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga
     4321 aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg
     4381 atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac
     4441 ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga
     4501 gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac
     4561 tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat
     4621 tagccatgtt taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa
     4681 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     4741 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     4801 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4861 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4921 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4981 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     5041 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     5101 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     5161 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     5221 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     5281 acctcgatcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     5341 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     5401 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     5461 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
     5521 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     5581 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     5641 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     5701 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     5761 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     5821 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5881 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5941 tcagaccctt ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa
     6001 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6061 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6121 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6181 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6241 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6301 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6361 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6421 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6481 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6541 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6601 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6661 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6721 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6781 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6841 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6901 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6961 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7021 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7081 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7141 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7201 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7261 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7321 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7381 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7441 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7501 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7561 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7621 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7681 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7741 ataaacaaat aggggttcc
//
