LOCUS       sequence-362054-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Fbxw7-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-sgFbxw7#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1693..1926
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2111..2155
                     /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    2453..2570
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2652..2900
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2652..2672
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2823..2842
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2922..2997
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3050..3549
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3085..3108)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3482..3501
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3553..3573
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3574..4602
                     /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    4658..5246
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4711..4731)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5129..5140)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5249..5265)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5250..5266)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5775..5955
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6017..6605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6097..6116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6776..7567,7568..7636))
                     /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     7399..7418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7637..7741)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg
     1561 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     1621 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     1681 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     1741 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     1801 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     1861 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     1921 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     1981 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     2041 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     2101 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     2161 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     2221 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     2281 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga
     2341 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg
     2401 atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa
     2461 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     2521 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     2581 acagggacag cagagatcca gtttggttag taccgggccc gctctagccc tgcaggaact
     2641 gtttaaactg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2701 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2761 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2821 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2881 tgtggaaagg accaaacacc gtgaacatgg tacaaggcca ggttttagag ctagaaatag
     2941 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     3001 tttgtttggc gcgcctatgg cttaattaac atggcggccg cattctaccg ggtaggggag
     3061 gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc
     3121 tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct
     3181 ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc
     3241 cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt
     3301 ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc
     3361 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg
     3421 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg
     3481 gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg
     3541 ggacccggga tgcccaagaa gaagaggaag gtgtccaatt tactgaccgt acaccaaaat
     3601 ttgcctgcat taccggtcga tgcaacgagt gatgaggttc gcaagaacct gatggacatg
     3661 ttcagggatc gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg
     3721 tcgtgggcgg catggtgcaa gttgaataac cggaaatggt ttcccgcaga acctgaagat
     3781 gttcgcgatt atcttctata tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa
     3841 catttgggcc agctaaacat gcttcatcgt cggtccgggc tgccacgacc aagtgacagc
     3901 aatgctgttt cactggttat gcggcggatc cgaaaagaaa acgttgatgc cggtgaacgt
     3961 gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc aggttcgttc actcatggaa
     4021 aatagcgatc gctgccagga tatacgtaat ctggcatttc tggggattgc ttataacacc
     4081 ctgttacgta tagccgaaat tgccaggatc agggttaaag atatctcacg tactgacggt
     4141 gggagaatgt taatccatat tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag
     4201 aaggcactta gcctgggggt aactaaactg gtcgagcgat ggatttccgt ctctggtgta
     4261 gctgatgatc cgaataacta cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca
     4321 tctgccacca gccagctatc aactcgcgcc ctggaaggga tttttgaagc aactcatcga
     4381 ttgatttacg gcgctaagga tgactctggt cagagatacc tggcctggtc tggacacagt
     4441 gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag tttcaatacc ggagatcatg
     4501 caagctggtg gctggaccaa tgtaaatatt gtcatgaact atatccgtaa cctggatagt
     4561 gaaacagggg caatggtgcg cctgctggaa gatggcgatt agccatgttt aagggttccg
     4621 gttccactag gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     4681 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     4741 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     4801 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     4861 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     4921 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     4981 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     5041 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     5101 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     5161 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     5221 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa
     5281 aacatggagc aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag
     5341 aagcacaaga ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa
     5401 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     5461 ggctaattca ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag
     5521 gctacttccc tgattggcag aactacacac cagggccagg gatcagatat ccactgacct
     5581 ttggatggtg ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag
     5641 gagagaacac ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag
     5701 aagtattaga gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc
     5761 atccggactg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     5821 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     5881 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     5941 ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     6001 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6061 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6121 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6181 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6241 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6301 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6361 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6421 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6481 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6541 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6601 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6661 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6721 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6781 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6841 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6901 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6961 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     7021 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7081 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7141 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7201 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7261 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7321 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7381 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7441 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7501 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7561 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7621 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7681 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7741 gcacatttcc ccgaaaagtg
//
