LOCUS       sequence-363662-        7748 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 7748)
  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 7748)
  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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(112..131)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        303..682
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        684..882
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     837..857
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             900..1080
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1127..1252
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1749..1982
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2167..2211
                     /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    2509..2626
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2708..2956
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2708..2728
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2879..2898
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2978..3053
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3106..3593
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3141..3164)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3526..3545
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3597..3617
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3618..4646
                     /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    4702..5290
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4755..4775)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5173..5184)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5293..5309)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5294..5310)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5819..5999
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6061..6649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6141..6160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6820..7611,7612..7680))
                     /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     7443..7462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7681..7748,1..37))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
       61 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
      121 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      181 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      241 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      301 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      361 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      421 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      481 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      541 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      601 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      661 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      721 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      781 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      841 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
      901 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
      961 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     1021 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     1081 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     1141 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     1201 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     1261 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     1321 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     1381 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     1441 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     1501 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     1561 aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag acctggagga
     1621 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa
     1681 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca
     1741 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca
     1801 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag
     1861 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc
     1921 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc
     1981 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct
     2041 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac
     2101 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag
     2161 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg
     2221 tttaacataa caaattggct gtggtatata aaattattca taatgatagt aggaggcttg
     2281 gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat
     2341 tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga
     2401 atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg
     2461 gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa
     2521 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     2581 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     2641 ggacagcaga gatccagttt ggttagtacc gggcccgctc tagccctgca ggaactgttt
     2701 aaactgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     2761 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     2821 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     2881 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     2941 gaaaggacca aacaccgaga cgggcataga tcacatggtt ttagagctag aaatagcaag
     3001 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     3061 tttggcgcgc ctatggctta attaacatgg cggccgcatt ctaccgggta ggggaggcgc
     3121 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     3181 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     3241 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     3301 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     3361 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     3421 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     3481 ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     3541 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggaccc gggatgccca
     3601 agaagaagag gaaggtgtcc aatttactga ccgtacacca aaatttgcct gcattaccgg
     3661 tcgatgcaac gagtgatgag gttcgcaaga acctgatgga catgttcagg gatcgccagg
     3721 cgttttctga gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt
     3781 gcaagttgaa taaccggaaa tggtttcccg cagaacctga agatgttcgc gattatcttc
     3841 tatatcttca ggcgcgcggt ctggcagtaa aaactatcca gcaacatttg ggccagctaa
     3901 acatgcttca tcgtcggtcc gggctgccac gaccaagtga cagcaatgct gtttcactgg
     3961 ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa caggctctag
     4021 cgttcgaacg cactgatttc gaccaggttc gttcactcat ggaaaatagc gatcgctgcc
     4081 aggatatacg taatctggca tttctgggga ttgcttataa caccctgtta cgtatagccg
     4141 aaattgccag gatcagggtt aaagatatct cacgtactga cggtgggaga atgttaatcc
     4201 atattggcag aacgaaaacg ctggttagca ccgcaggtgt agagaaggca cttagcctgg
     4261 gggtaactaa actggtcgag cgatggattt ccgtctctgg tgtagctgat gatccgaata
     4321 actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc accagccagc
     4381 tatcaactcg cgccctggaa gggatttttg aagcaactca tcgattgatt tacggcgcta
     4441 aggatgactc tggtcagaga tacctggcct ggtctggaca cagtgcccgt gtcggagccg
     4501 cgcgagatat ggcccgcgct ggagtttcaa taccggagat catgcaagct ggtggctgga
     4561 ccaatgtaaa tattgtcatg aactatatcc gtaacctgga tagtgaaaca ggggcaatgg
     4621 tgcgcctgct ggaagatggc gattagccat gtttaagggt tccggttcca ctaggtacaa
     4681 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     4741 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4801 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4861 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4921 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4981 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5041 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     5101 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5161 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5221 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5281 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     5341 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     5401 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     5461 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5521 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     5581 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     5641 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     5701 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     5761 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     5821 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5881 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5941 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     6001 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6061 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6121 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6181 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6241 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6301 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6361 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6421 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6481 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6541 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6601 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6661 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6721 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6781 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6841 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6901 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6961 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7021 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7081 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7141 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7201 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7261 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7321 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7381 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7441 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7501 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7561 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7621 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7681 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7741 catatttg
//
