LOCUS       sequence-363592-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Smad4-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-sgSmad4#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(66..85)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        257..636
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        638..836
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     791..811
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             854..1034
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1081..1206
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1703..1936
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2121..2165
                     /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    2463..2580
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2662..2910
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2662..2682
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2833..2852
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2931..3006
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3059..3558
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3094..3117)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3491..3510
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3562..3582
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3583..4611
                     /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    4667..5255
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4720..4740)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5138..5149)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5258..5274)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5259..5275)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5784..5964
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6026..6614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6106..6125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6785..7576,7577..7645))
                     /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     7408..7427
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7646..7750)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
       61 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
      121 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
      181 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
      241 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
      301 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      361 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      421 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      481 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      541 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      601 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      661 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      721 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      781 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg
      841 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      901 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      961 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     1021 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     1081 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     1141 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     1201 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     1261 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     1321 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     1381 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     1441 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1501 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgctgatct
     1561 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1621 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1681 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1741 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1801 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1861 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1921 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     1981 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     2041 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     2101 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     2161 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     2221 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     2281 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg
     2341 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat
     2401 tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt attcatccac
     2461 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     2521 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     2581 cgggtttatt acagggacag cagagatcca gtttggttag taccgggccc gctctagccc
     2641 tgcaggaact gtttaaactg tgagggccta tttcccatga ttccttcata tttgcatata
     2701 cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta
     2761 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2821 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2881 ttatatatct tgtggaaagg accaaacacc gatgtgtcat agacaaggtg gttttagagc
     2941 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     3001 cggtgctttt ttgtttggcg cgcctatggc ttaattaaca tggcggccgc attctaccgg
     3061 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc
     3121 acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
     3181 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
     3241 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
     3301 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
     3361 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     3421 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     3481 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     3541 tcatctccgg gacccgggat gcccaagaag aagaggaagg tgtccaattt actgaccgta
     3601 caccaaaatt tgcctgcatt accggtcgat gcaacgagtg atgaggttcg caagaacctg
     3661 atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat gcttctgtcc
     3721 gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa
     3781 cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc agtaaaaact
     3841 atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct gccacgacca
     3901 agtgacagca atgctgtttc actggttatg cggcggatcc gaaaagaaaa cgttgatgcc
     3961 ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca ggttcgttca
     4021 ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct ggggattgct
     4081 tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga tatctcacgt
     4141 actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt tagcaccgca
     4201 ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg gatttccgtc
     4261 tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa aaatggtgtt
     4321 gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat ttttgaagca
     4381 actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct ggcctggtct
     4441 ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt ttcaataccg
     4501 gagatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta tatccgtaac
     4561 ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgatta gccatgttta
     4621 agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga
     4681 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     4741 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     4801 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     4861 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     4921 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     4981 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     5041 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     5101 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     5161 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     5221 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag
     5281 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
     5341 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
     5401 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     5461 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
     5521 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
     5581 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
     5641 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
     5701 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
     5761 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
     5821 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     5881 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     5941 agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc
     6001 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6061 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6121 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6181 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6241 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6301 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6361 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6421 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6481 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6541 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6601 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6661 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6721 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6781 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6841 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6901 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6961 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7021 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7081 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7141 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7201 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7261 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7321 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7381 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7441 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7501 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7561 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7621 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7681 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7741 gggttccgcg cacatttcc
//
