LOCUS       sequence-363618-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Ubr5-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-sgUbr5#1/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(134..153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        325..704
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        706..904
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     859..879
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             922..1102
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1149..1274
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1771..2004
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2189..2233
                     /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    2531..2648
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2730..2978
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2730..2750
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2901..2920
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3000..3075
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3128..3615
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3163..3186)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3548..3567
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3619..3639
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3640..4668
                     /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    4724..5312
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4777..4797)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5195..5206)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5315..5331)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5316..5332)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5841..6021
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6083..6671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6163..6182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6842..7633,7634..7702))
                     /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     7465..7484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7703..7748,1..59))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
       61 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
      121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      181 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      241 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      301 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      361 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      421 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      481 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      541 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      601 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      661 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      721 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      781 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      841 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      901 gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
      961 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     1021 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     1081 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
     1141 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     1201 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     1261 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
     1321 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
     1381 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     1441 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1501 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1561 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc
     1621 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1681 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1741 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1801 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1861 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1921 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1981 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     2041 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     2101 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     2161 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     2221 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     2281 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     2341 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     2401 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     2461 cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat
     2521 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2581 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2641 aaaattttcg ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc
     2701 tctagccctg caggaactgt ttaaactgtg agggcctatt tcccatgatt ccttcatatt
     2761 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     2821 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2881 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2941 tcttggcttt atatatcttg tggaaaggac caaacaccga gtgaatctta cctgccgggg
     3001 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     3061 gcaccgagtc ggtgcttttt tgtttggcgc gcctatggct taattaacat ggcggccgca
     3121 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     3181 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     3241 ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     3301 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     3361 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     3421 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg
     3481 ggaaggggtg ggtccggggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     3541 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     3601 tctccgggac ccgggatgcc caagaagaag aggaaggtgt ccaatttact gaccgtacac
     3661 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg
     3721 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt
     3781 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct
     3841 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc
     3901 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt
     3961 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt
     4021 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc
     4081 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat
     4141 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact
     4201 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt
     4261 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct
     4321 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc
     4381 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact
     4441 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga
     4501 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag
     4561 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg
     4621 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagcc atgtttaagg
     4681 gttccggttc cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta
     4741 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4801 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4861 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4921 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4981 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     5041 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5101 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     5161 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5221 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5281 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc
     5341 tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct
     5401 ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa
     5461 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5521 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
     5581 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
     5641 tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca
     5701 atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     5761 agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
     5821 agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     5881 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5941 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     6001 cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6061 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6121 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6181 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6241 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6301 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6361 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6421 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6481 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6541 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6601 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6661 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6721 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6781 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6841 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6901 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6961 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7021 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7081 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7141 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7201 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7261 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7321 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7381 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7441 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7501 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7561 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7621 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7681 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7741 gttattgt
//
