LOCUS       sequence-363664-        7760 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 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-sgRnf43#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(142..161)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        333..712
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        714..912
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     867..887
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             930..1110
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1157..1282
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1779..2012
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2197..2241
                     /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    2539..2656
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2738..2986
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2738..2758
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2909..2928
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3008..3083
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3136..3635
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3171..3194)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3568..3587
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3639..3659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3660..4688
                     /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    4744..5332
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4797..4817)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5215..5226)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5335..5351)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5336..5352)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5861..6041
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6103..6691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6183..6202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6862..7653,7654..7722))
                     /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     7485..7504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7723..7760,1..67))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
       61 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
      121 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
      181 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
      241 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
      301 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
      361 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      421 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      481 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      541 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      601 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      661 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
      721 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
      781 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
      841 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
      901 ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct
      961 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     1021 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     1081 ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa
     1141 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag
     1201 aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga
     1261 gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa
     1321 aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag
     1381 cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag
     1441 acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt
     1501 atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa
     1561 ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc
     1621 ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     1681 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     1741 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     1801 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     1861 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     1921 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     1981 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     2041 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     2101 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     2161 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     2221 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     2281 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     2341 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     2401 accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga
     2461 gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat
     2521 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2581 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2641 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc
     2701 gggcccgctc tagccctgca ggaactgttt aaactgtgag ggcctatttc ccatgattcc
     2761 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     2821 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     2881 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     2941 tttcgatttc ttggctttat atatcttgtg gaaaggacca aacaccgtag acagatggca
     3001 cacacgggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     3061 aaaaagtggc accgagtcgg tgcttttttg tttggcgcgc ctatggctta attaacatgg
     3121 cggccgcatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt
     3181 tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca
     3241 catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta
     3301 ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac
     3361 aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga
     3421 agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc
     3481 agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg
     3541 gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg
     3601 cgctgttctc ctcttcctca tctccgggac ccgggatgcc caagaagaag aggaaggtgt
     3661 ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca acgagtgatg
     3721 aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct gagcatacct
     3781 ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg aataaccgga
     3841 aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt caggcgcgcg
     3901 gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt catcgtcggt
     3961 ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg cggatccgaa
     4021 aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt
     4081 tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata cgtaatctgg
     4141 catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc aggatcaggg
     4201 ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc agaacgaaaa
     4261 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg
     4321 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg
     4381 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg
     4441 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga
     4501 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg
     4561 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca
     4621 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg
     4681 gcgattagcc atgtttaagg gttccggttc cactaggtac aattcgatat caagcttatc
     4741 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     4801 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     4861 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     4921 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     4981 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     5041 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     5101 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     5161 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     5221 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     5281 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
     5341 cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca atacagcagc
     5401 taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg gttttccagt
     5461 cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     5521 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatatcct
     5581 tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact acacaccagg
     5641 gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac cagttgagca
     5701 agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc ctgtgagcct
     5761 gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca gccgcctagc
     5821 atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct ggttagacca
     5881 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5941 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     6001 atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag caaaaggcca
     6061 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6121 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6181 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6241 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6301 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6361 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6421 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6481 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6541 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6601 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6661 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6721 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6781 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6841 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6901 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6961 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     7021 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     7081 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     7141 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     7201 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     7261 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     7321 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     7381 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     7441 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7501 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7561 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7621 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7681 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7741 ttattgaagc atttatcagg
//
