LOCUS       sequence-180653-        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   Rogers ZN, McFarland CD, Winters IP, Naranjo S, Chuang CH, Petrov D,
            Winslow MM
  TITLE     A quantitative and multiplexed approach to uncover the fitness 
            landscape of tumor suppression in vivo.
  JOURNAL   Nat Methods. 2017 May 22. doi: 10.1038/nmeth.4297.
  PUBMED    28530655
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: "Nat 
            Methods. 2017 May 22. doi: 10.1038/nmeth.4297."
COMMENT     Sequence Label: Lenti-sgSmad4/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2643..2891
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2643..2663
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2814..2833
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2912..2987
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3040..3539
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3075..3098)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3472..3491
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3543..3563
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3564..4592
                     /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    4648..5236
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4701..4721)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5119..5130)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5239..5255)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5240..5256)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5765..5945
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6007..6595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6087..6106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6766..7557,7558..7626))
                     /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     7389..7408
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7627..7731)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagcc ctgcaggaac tgtttaaact
     2641 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2701 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2761 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     2821 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     2881 gaccaaacac cggtggcgtt agactctgcc ggttttagag ctagaaatag caagttaaaa
     2941 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgtttggc
     3001 gcgcctatgg cttaattaac atggcggccg cattctaccg ggtaggggag gcgcttttcc
     3061 caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg
     3121 gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg
     3181 gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc
     3241 agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga
     3301 tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc
     3361 tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca
     3421 ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca
     3481 cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggacccggga
     3541 tgcccaagaa gaagaggaag gtgtccaatt tactgaccgt acaccaaaat ttgcctgcat
     3601 taccggtcga tgcaacgagt gatgaggttc gcaagaacct gatggacatg ttcagggatc
     3661 gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg
     3721 catggtgcaa gttgaataac cggaaatggt ttcccgcaga acctgaagat gttcgcgatt
     3781 atcttctata tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa catttgggcc
     3841 agctaaacat gcttcatcgt cggtccgggc tgccacgacc aagtgacagc aatgctgttt
     3901 cactggttat gcggcggatc cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg
     3961 ctctagcgtt cgaacgcact gatttcgacc aggttcgttc actcatggaa aatagcgatc
     4021 gctgccagga tatacgtaat ctggcatttc tggggattgc ttataacacc ctgttacgta
     4081 tagccgaaat tgccaggatc agggttaaag atatctcacg tactgacggt gggagaatgt
     4141 taatccatat tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag aaggcactta
     4201 gcctgggggt aactaaactg gtcgagcgat ggatttccgt ctctggtgta gctgatgatc
     4261 cgaataacta cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca
     4321 gccagctatc aactcgcgcc ctggaaggga tttttgaagc aactcatcga ttgatttacg
     4381 gcgctaagga tgactctggt cagagatacc tggcctggtc tggacacagt gcccgtgtcg
     4441 gagccgcgcg agatatggcc cgcgctggag tttcaatacc ggagatcatg caagctggtg
     4501 gctggaccaa tgtaaatatt gtcatgaact atatccgtaa cctggatagt gaaacagggg
     4561 caatggtgcg cctgctggaa gatggcgatt agccatgttt aagggttccg gttccactag
     4621 gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     4681 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     4741 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     4801 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     4861 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     4921 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     4981 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     5041 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     5101 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     5161 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     5221 tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc
     5281 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     5341 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
     5401 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     5461 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
     5521 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
     5581 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
     5641 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
     5701 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
     5761 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5821 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5881 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5941 tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6001 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6061 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6121 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6181 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6241 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6301 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6361 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6421 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6481 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6541 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6601 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6661 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6721 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6781 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6841 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6901 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6961 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7021 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7081 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7141 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7201 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7261 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7321 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7381 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7441 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7501 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7561 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7621 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7681 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7741 ccgaaaagtg ccacctgac
//
