LOCUS       sequence-363559-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Wrn-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-sgWrn#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(137..156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        328..707
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        709..907
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     862..882
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             925..1105
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1152..1277
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1774..2007
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2192..2236
                     /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    2534..2651
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2733..2981
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2733..2753
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2904..2923
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3002..3077
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3130..3629
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3165..3188)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3562..3581
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3633..3653
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3654..4682
                     /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    4738..5326
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4791..4811)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5209..5220)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5329..5345)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5330..5346)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5855..6035
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6097..6685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6177..6196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6856..7647,7648..7716))
                     /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     7479..7498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7717..7759,1..62))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
       61 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
      121 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      181 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      241 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      301 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      361 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      421 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      481 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      541 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      601 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      661 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      721 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      781 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      841 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      901 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
      961 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1021 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1081 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1141 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1201 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1261 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1321 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1381 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1441 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1501 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1561 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1621 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1681 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1741 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1801 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1861 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1921 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1981 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     2041 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     2101 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     2161 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     2221 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     2281 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     2341 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2401 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2461 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag
     2521 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2581 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2641 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc
     2701 cgctctagcc ctgcaggaac tgtttaaact gtgagggcct atttcccatg attccttcat
     2761 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     2821 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     2881 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2941 atttcttggc tttatatatc ttgtggaaag gaccaaacac cgcgtctgtc tgatggcgat
     3001 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     3061 tggcaccgag tcggtgcttt tttgtttggc gcgcctatgg cttaattaac atggcggccg
     3121 cattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag
     3181 ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca
     3241 ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc
     3301 ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg
     3361 aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg
     3421 taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
     3481 tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc
     3541 gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt
     3601 tctcctcttc ctcatctccg ggacccggga tgcccaagaa gaagaggaag gtgtccaatt
     3661 tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc
     3721 gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa
     3781 tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt
     3841 ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg
     3901 cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc
     3961 tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa
     4021 acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc
     4081 aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc
     4141 tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag
     4201 atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg
     4261 ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat
     4321 ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa
     4381 aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga
     4441 tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc
     4501 tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag
     4561 tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact
     4621 atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt
     4681 agccatgttt aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat
     4741 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4801 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4861 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4921 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4981 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     5041 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     5101 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     5161 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     5221 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5281 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     5341 cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
     5401 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
     5461 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5521 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5581 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg
     5641 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa
     5701 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg
     5761 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca
     5821 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg
     5881 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     5941 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     6001 cagacccttt tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa
     6061 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6121 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6181 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6241 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6301 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6361 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6421 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6481 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6541 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6601 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6661 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7741 aagcatttat cagggttat
//
