LOCUS       sequence-362056-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Brca2-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-sgBrca2#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2..106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(181..200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        372..751
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        753..951
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     906..926
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             969..1149
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1196..1321
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1818..2051
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2236..2280
                     /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    2578..2695
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2777..3025
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2777..2797
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2948..2967
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3046..3121
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3174..3673
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3209..3232)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3606..3625
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3677..3697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3698..4726
                     /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    4782..5370
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4835..4855)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5253..5264)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5373..5389)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5374..5390)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5899..6079
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6141..6729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6221..6240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6900..7691,7692..7759,1))
                     /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     7523..7542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
       61 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      121 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
      181 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      241 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
      301 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      361 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      421 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      481 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      541 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      601 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      661 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      721 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      781 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      841 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      901 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg
      961 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     1021 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     1081 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     1141 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     1201 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     1261 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     1321 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     1381 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     1441 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     1501 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1561 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1621 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct gatcttcaga
     1681 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     1741 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     1801 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     1861 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     1921 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     1981 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     2041 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     2101 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     2161 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     2221 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     2281 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     2341 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     2401 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     2461 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     2521 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     2581 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2641 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2701 ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct agccctgcag
     2761 gaactgttta aactgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2821 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2881 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2941 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     3001 tatcttgtgg aaaggaccaa acaccgtacc caaagtctcg tcaaggtttt agagctagaa
     3061 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     3121 cttttttgtt tggcgcgcct atggcttaat taacatggcg gccgcattct accgggtagg
     3181 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     3241 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     3301 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     3361 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     3421 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     3481 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     3541 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3601 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3661 tccgggaccc gggatgccca agaagaagag gaaggtgtcc aatttactga ccgtacacca
     3721 aaatttgcct gcattaccgg tcgatgcaac gagtgatgag gttcgcaaga acctgatgga
     3781 catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc tgtccgtttg
     3841 ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg cagaacctga
     3901 agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa aaactatcca
     3961 gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac gaccaagtga
     4021 cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga
     4081 acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc gttcactcat
     4141 ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga ttgcttataa
     4201 caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatatct cacgtactga
     4261 cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca ccgcaggtgt
     4321 agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt ccgtctctgg
     4381 tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc
     4441 gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg aagcaactca
     4501 tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct ggtctggaca
     4561 cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa taccggagat
     4621 catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc gtaacctgga
     4681 tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gattagccat gtttaagggt
     4741 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca
     4801 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     4861 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     4921 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     4981 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     5041 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     5101 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     5161 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     5221 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     5281 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     5341 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta
     5401 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg
     5461 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga
     5521 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     5581 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca
     5641 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg
     5701 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat
     5761 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag
     5821 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag
     5881 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5941 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     6001 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     6061 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6121 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6181 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6241 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6301 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6361 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6421 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6481 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6541 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6601 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6661 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6721 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6781 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6841 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6901 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6961 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7021 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7081 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7141 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7201 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7261 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7321 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7381 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7441 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7501 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7561 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7621 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7681 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7741 cggaaatgtt gaatactca
//
