LOCUS       sequence-363657-        7760 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 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-sgWrn#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(118..137)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        309..688
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        690..888
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     843..863
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             906..1086
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1133..1258
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1755..1988
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2173..2217
                     /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    2515..2632
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2714..2962
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2714..2734
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2885..2904
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2984..3059
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3112..3611
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3147..3170)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3544..3563
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3615..3635
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3636..4664
                     /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    4720..5308
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4773..4793)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5191..5202)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5311..5327)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5312..5328)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5837..6017
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6079..6667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6159..6178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6838..7629,7630..7698))
                     /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     7461..7480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7699..7760,1..43))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
       61 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
      121 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
      181 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
      241 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      301 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      361 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      421 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      481 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      541 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      601 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      661 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      721 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      781 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      841 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct
      901 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
      961 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     1021 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     1081 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
     1141 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     1201 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     1261 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     1321 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     1381 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     1441 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     1501 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     1561 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct
     1621 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1681 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1741 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1801 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1861 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1921 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1981 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     2041 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2101 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2161 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2221 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2281 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2341 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2401 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2461 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2521 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2581 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2641 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctagc cctgcaggaa
     2701 ctgtttaaac tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2761 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2821 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2881 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2941 cttgtggaaa ggaccaaaca ccgaaaccta aatagtgacg tgggttttag agctagaaat
     3001 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     3061 tttttgtttg gcgcgcctat ggcttaatta acatggcggc cgcattctac cgggtagggg
     3121 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     3181 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     3241 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3301 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     3361 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     3421 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     3481 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     3541 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     3601 cgggacccgg gatgcccaag aagaagagga aggtgtccaa tttactgacc gtacaccaaa
     3661 atttgcctgc attaccggtc gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca
     3721 tgttcaggga tcgccaggcg ttttctgagc atacctggaa aatgcttctg tccgtttgcc
     3781 ggtcgtgggc ggcatggtgc aagttgaata accggaaatg gtttcccgca gaacctgaag
     3841 atgttcgcga ttatcttcta tatcttcagg cgcgcggtct ggcagtaaaa actatccagc
     3901 aacatttggg ccagctaaac atgcttcatc gtcggtccgg gctgccacga ccaagtgaca
     3961 gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga aaacgttgat gccggtgaac
     4021 gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg
     4081 aaaatagcga tcgctgccag gatatacgta atctggcatt tctggggatt gcttataaca
     4141 ccctgttacg tatagccgaa attgccagga tcagggttaa agatatctca cgtactgacg
     4201 gtgggagaat gttaatccat attggcagaa cgaaaacgct ggttagcacc gcaggtgtag
     4261 agaaggcact tagcctgggg gtaactaaac tggtcgagcg atggatttcc gtctctggtg
     4321 tagctgatga tccgaataac tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc
     4381 catctgccac cagccagcta tcaactcgcg ccctggaagg gatttttgaa gcaactcatc
     4441 gattgattta cggcgctaag gatgactctg gtcagagata cctggcctgg tctggacaca
     4501 gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg agtttcaata ccggagatca
     4561 tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa ctatatccgt aacctggata
     4621 gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga ttagccatgt ttaagggttc
     4681 cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa
     4741 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     4801 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     4861 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     4921 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     4981 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     5041 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     5101 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     5161 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     5221 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     5281 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga
     5341 aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct
     5401 agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc
     5461 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     5521 agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca
     5581 aggctacttc cctgattggc agaactacac accagggcca gggatcagat atccactgac
     5641 ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga
     5701 aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag
     5761 agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct
     5821 gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     5881 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     5941 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     6001 gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6061 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6121 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6421 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6481 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6541 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6601 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6661 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6721 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6781 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6901 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6961 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     7021 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7081 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7141 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7201 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7261 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7321 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7381 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7441 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7501 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7561 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7621 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7681 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7741 ttgtctcatg agcggataca
//
